Hematopoietic stem cell transplant-associated thrombotic microangiopathy: current paradigm and novel therapies.

Hematopoietic stem cell transplantation-associated thrombotic microangiopathy (TA-TMA) remains a difficult complication to address due to its high mortality rate, lack of standard diagnostic criteria and limited therapeutic options. Underscoring this challenge is the complex pathophysiology involved and multiple contributing factors that converge on a final pathway involving widespread endothelial injury and complement activation. In addressing our current understanding of TA-TMA, we highlight the risk factors leading to endothelial damage and a pathophysiological cascade that ensues. We have also compared the different definition criteria and biomarkers that can enable early intervention in TA-TMA patients. Current first-line management includes discontinuation or alteration of the immunosuppressive regimen, treatment of co-existing infectious and GVHD, aggressive hypertension control and supportive therapy. We discuss current pharmacological therapies, including newer agents that target the complement cascade and nitric oxide pathways.

A prospective study of the incidence and open-label treatment of interferon-induced major depressive disorder in patients with hepatitis C.

Interferon (IFN) therapy has been associated with the development of Major Depressive Disorder (MDD) when given to patients with hepatitis C (HCV). The incidence, time course, risk factors, and treatment of IFN-induced MDD are poorly understood. The objectives of the present study were to determine the incidence of IFN-induced MDD, as well as to determine the efficacy of open-label antidepressant treatment, in particular selective serotonin reuptake inhibitors (SSRIs) for IFN-induced MDD. Thirty-nine HCV patients on IFN therapy were monitored weekly using the Beck Depression Inventory (BDI). Those who became depressed were treated with citalopram, a SSRI antidepressant. Main outcome measures included the incidence of IFN-induced MDD, as well as response rates to antidepressants in those patients who developed IFN-induced MDD. Our results showed that 13 of 39 patients (33%) developed IFN-induced MDD. There were no differences in age, gender, past history of MDD, or substance use between those who became depressed and those who did not. However, there were significantly fewer African American patients in the depressed group. Patients who developed IFN-induced MDD were on IFN therapy for an average of 12.1 weeks prior to the development of MDD. Eleven of 13 patients (85%) were responsive to antidepressant treatment. We conclude that IFN-induced MDD is common in HCV patients. Health care providers should follow IFN-treated HCV patients for the development of MDD, particularly between the 2nd and 5th months of IFN therapy. SSRIs, in particular citalopram, are an effective treatment for IFN-induced depression in HCV patients.

Transforming growth factor-beta(1) modifies fibroblast growth factor-2 production in type II cells.

Transforming growth factor (TGF)-beta(1) is an inflammatory cytokine that plays multiple roles in pulmonary fibrosis. In vascular epithelium, it has been shown to regulate production and activity of fibroblast growth factor (FGF)-2, a potent type II cell mitogen in the lung. Such a relationship could have important consequences in prefibrotic change in the lung alveolus, where reepithelialization of alveolar surfaces is crucial. The goal of this study was to determine if FGF-2 production by alveolar type II cells is modulated by TGF-beta(1) or FGF-1, another type II cell mitogen. Isolated rat type II cells were exposed to 0 to 40 ng/mL of TGF-beta(1) or 0 to 500 ng/mL of FGF-1 in serum-free medium for 1 to 3 days. Using a specific immunoassay, significant increases in FGF-2 protein in type II cell lysates were achieved after 1 day of exposure to 100 ng/mL of FGF-1 and after 3 days of treatment with 8 ng/mL of TGF-beta(1). Similarly, transcripts for FGF-2 were dramatically increased with TGF-beta(1) or FGF-1, as were those for FGF receptor (FGFR)-1. These interactions were dramatically effected by the addition of heparin, a model sulfated extracellular matrix (ECM). Heparin as low as 0.01 mg/mL significantly downregulated expression of TGF-beta(1) and FGF-1-stimulated FGF-2 and FGFR-1. These results demonstrate important regulatory links between FGF-2, sulfated ECMs, and both TGF-beta(1) and FGF-1, which could contribute to the modulation of normal cell turnover, development, and repair processes attendant to fibrosis in the lung.

TGF-beta1 and fibroblast growth factor-1 modify fibroblast growth factor-2 production in type II cells.

Fibroblast growth factor (FGF)-2, which stimulates DNA synthesis by type II cells in the lung, has been shown to be regulated by transforming growth factor (TGF)-beta1, an important inflammatory cytokine, in vascular epithelium. The goal of this study was to determine if FGF-2 production by alveolar type II cells is modulated by TGF-beta1 or FGF-1, which also stimulates DNA synthesis by type II cells. Isolated rat type II cells were exposed to 0-40 ng/ml of TGF-beta1 or 0-500 ng/ml of FGF-1 in serum-free medium for 1-5 days. With a specific immunoassay, significant increases of FGF-2 protein in type II cell lysates to levels above those in control cells were achieved after 1 day of exposure to 100 ng/ml of FGF-1 and after 3 days of treatment with 8 ng/ml of TGF-beta1. Similarly, transcripts for FGF-2 were dramatically increased above those in control cells with TGF-beta1 or FGF-1, as were those for FGF receptor-1. These results demonstrate important regulatory links between FGF-2 and both TGF-beta1 and FGF-1 in the alveolar epithelium that could contribute to the regulation of normal cell turnover, development, and the repair processes after injury in the lung.

Sulfation of extracellular matrices modifies growth factor effects on type II cells on laminin substrata.

The alveolar basement membrane contains a variety of extracellular matrix (ECM) molecules, including laminin and sulfated glycosaminoglycans of proteoglycans. These mixtures exist within microdomains of differing levels of sulfate, which may specifically interact to be key determinants of the known capacity of the type II cell to respond to certain growth factors. Isolated type II cells were exposed to either acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), or keratinocyte growth factor (KGF; FGF-7) on culture wells precoated with laminin alone or in combination with chondroitin sulfate (CS), high-molecular-weight heparin, or their desulfated forms. Desulfated heparin significantly elevated FGF-1- and FGF-2-stimulated DNA synthesis, whereas desulfated CS and N-desulfated heparin elevated FGF-7-stimulated DNA synthesis by type II cells on laminin substrata. When FGF-1 was mixed into the various test matrix substrata, DNA synthesis was significantly increased in all cases. These results demonstrated that decreased levels of sulfate in ECM substrata act to upregulate responses to heparin-binding growth factors by alveolar epithelial cells on laminin substrata.

Biosynthesis of sulfated extracellular matrices by alveolar type II cells increases with time in culture.

The aim of this study was to determine the extent to which sulfate incorporated into biosynthesized basement membrane (BM) components increased as isolated type II cells progress toward a more type I cell-like phenotype from 7 to 21 days in culture. Specific sulfate cytochemistry, using high iron diamine, showed that type I-like cells in 21-day cultures deposited a more highly sulfated extracellular matrix. Biosynthetic labeling experiments using [35S]cysteine or [35S]sulfate as precursors confirmed the increased capacity of 21-day type I-like cells to biosynthesize sulfated BM components compared with type II-like cells in 7-day cultures, including a novel sulfated laminin. These biochemical changes in sulfation of BM components coincide with the established phenotypic transition from type II to type I cells during prolonged culture. More importantly, the data suggest that regulation of sulfation constitutes a potential mechanism by which type I and type II cells alter their environment in such a manner as to stabilize phenotype and modulate responses to growth factors.

Sulfation of extracellular matrices modifies responses of alveolar type II cells to fibroblast growth factors.

The pulmonary alveolar basement membrane (BM) associated with alveolar type II cells has been shown to be significantly less sulfated than that of type I cells. To examine the biological significance of this observation, we measured the incorporation of 5-bromodeoxyuridine (BrdU) as an indicator of DNA synthesis in isolated rat type II cells cultured for 72-120 h on substrata that were naturally sulfated, not sulfated, or chemically desulfated in serum-free, hormonally defined media, with and without selected growth factors. The percentage of cells incorporating BrdU was significantly elevated by desulfated chondroitin sulfate in the presence of fibroblast growth factor-2 (FGF-2 or basic FGF) and depressed by heparin in the presence of either FGF-1 or acidic FGF or FGF-2. This depressive effect was lost by removing sulfate from the heparin. Some responses were dependent on the period of time in culture and concentration and molecular weight of the substrata. These observations support the notion that sulfation per se of certain components of BM is a key determinant of type II cell responses to select growth factors that may define patterns of proliferation and differentiation.

Heterogeneity of sulfated microdomains within basement membranes of pulmonary airway epithelium.

The purpose of this study was to determine whether the cytochemically defined distribution of sulfated macromolecules is significantly different in microdomains of basement membranes (BMs) associated with different levels of pulmonary airways. The high iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) technique, which is highly specific for sulfate esters of glycosaminoglycans and some glycoproteins, was used as a probe to compare the BM of trachea, bronchi, and three different sizes of bronchioles. When HID-reactive sites were counted and statistically compared, significant differences were found between the three known anatomically distinct layers of the BM--lamina lucida (LL), lamina densa (LD), and lamina reticularis (LR)--relative to the airway level. The highest concentration of HID reactivity in trachea, bronchi, and large bronchiole was found in LR and the lowest in LD. By comparison, HID-reactive sites were found to be more concentrated in the LL in medium and small bronchioles. HID reactivity was consistently low in LD as compared with LL and LR in all five locations. The overall degree of HID reactivity in BMs was clearly highest in large bronchioles and lowest in medium and small bronchioles. This cytochemically detectable heterogeneity in the distribution of HID reactivity in BM microdomains may represent specific compositional differences in pulmonary BMs which are important determinants of epithelial cell function and might be expected to impact key biologic processes in normal and pathologic states.

Immunohistochemical localization of chondroitin sulfate, chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, entactin, and laminin in basement membranes of postnatal developing and adult rat lungs.

Histologic preparations of lungs from 1-, 5-, 10-, 18-, and 25-day-old postnatal and adult rats were examined immunohistochemically with antibodies specific against chondroitin sulfate (CS), basement membrane chondroitin sulfate proteoglycan (BM-CSPG), heparan sulfate proteoglycan (HSPG), entactin, and laminin. A monoclonal antibody specific for the glycosaminoglycan portion (CS) of CSPG and a monoclonal antibody against the core protein of CSPG were used in an immunoperoxidase sequence to stain extracellular matrix (ECM) components of pulmonary basement membranes (BMs). Anti-CS stained airway BM strongly and alveolar BM weakly in the adult rat lung, as well as in vascular and airway adventitia. In developing lungs, immunoreactivity was strong in all ECM sites, including BM, at day 1 postnatal, and progressively diminished thereafter except in vascular and airway adventitia. Anti-CSPG stained alveolar, airway, and vascular BMs, in addition to smooth muscle external laminae (EL), in the adult and developing rat. Immunostaining for CSPG required hyaluronidase digestion, whereas CS staining was lost with the same treatment. A polyclonal antibody to the core protein of HSPG was found to be similarly distributed to CSPG by immunoperoxidase staining in adult and developing rat lungs, with the notable exception that little immunoreactivity for HSPG was found in smooth muscle EL. Commercially obtained polyclonal antibodies to entactin and laminin gave immunostaining comparable to that seen with CSPG, except that entactin showed particular affinity for EL. These results offer a more detailed perspective on previous survey observations of CSPG, HSPG, and entactin in the rat lung, and describe the immunoreactivity of CS for the first time.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical localization of epidermal growth factor and acidic and basic fibroblast growth factors in postnatal developing and adult rat lungs.

Histologic preparations of lungs form 1-, 5-, 10-, 18-, and 25-day-old rats and adult rats were probed immunohistochemically with specific antibodies for the distribution of epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (bFGF). Immunoperoxidase staining of sections of adult rat lungs with a rabbit polyclonal antibody to bovine EGF was strong in ciliated cells of airways and mast cells, nonciliated cells of bronchioles, smooth muscle, type II cells of alveoli, and interstitial and epithelial cells of alveolar septal regions. Developing postnatal lungs had moderate and somewhat diffuse immunoreactivity in all epithelial cells, and more intense staining in vascular smooth muscle. Immunoperoxidase staining of adult rat lungs with a rabbit polyclonal antibody against bovine aFGF was distributed in identical fashion to EGF, with the exception of mast cells which were not reactive. These same sights were localized using an immunoperoxidase sequence with a rabbit polyclonal antibody to the leu 60-leu 98 fragment of aFGF (aFGFfr) with less background. In postnatal developing lungs, immunoreactivity with aFGF and aFGFfr preparations was diffuse and moderately intense in epithelial cells and vascular smooth muscle. Immunoperoxidase staining of adult rat lung sections with a monoclonal antibody to bovine bFGF was strong in hyaluronidase-digested preparations. Reactivity was principally confined to alveolar and vascular basement membrane regions and external laminae of smooth muscle. In early postnatal development, immunoreactivity for bFGF was found in basement membranes beneath developing epithelium and the endothelium of vessel wall intima and in the adventitia, with reactivity increasing with advancing age.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphologic and functional effects of bile salt on the porcine colon during injury and repair.

Deoxycholate-induced colonic injury and repair were studied both functionally and morphologically utilizing in vivo loop preparations of the porcine colon. The mucosa was exposed to (a) varying doses (1.5 to 21 mM) of deoxycholate for 30 minutes, (b) 15 mM deoxycholate for varying times and (c) 15 mM deoxycholate for 30 minutes with varying times of recovery. Colonic permeability was assessed by mannitol clearance from blood to lumen and transmural potential difference. After colonic perfusion, tissue samples were collected for light and electron microscopy. Both the degree of mucosal permeability and the amount of superficial epithelial damage increased with increasing concentrations of bile salt culminating in cell necrosis and epithelial sloughing. Denuded colonic surfaces became reepithelialized by migrating, flattened cells in as little as 15 minutes of recovery. Relatively normal appearing columnar epithelium was restituted within 2 hours. Mannitol clearance returned to control values after 30 minutes of recovery, whereas it took potential difference 2 hours of recovery to return to normal. The results of these experiments suggest that (a) the permeability changes measured are most likely due to the lytic action of bile salts which leads to cell degeneration and sloughing of the superficial epithelium, (b) epithelial restitution after superficial damage is remarkably rapid, (c) the formation of a flattened epithelium of immature cells is adequate for restoration of the barrier to macromolecules but ion transport or resistance is slower to recover and (d) repair is due to an active ameboid movement of viable cells out of the crypts onto the surface of the colon.