Jean-Alexis Grimaud

HEPARIN DECREASES THE BLOOD CLEARANCE OF INTERFERON-GAMMA AND INCREASES ITS ACTIVITY BY LIMITING THE PROCESSING OF ITS CARBOXYL-TERMINAL SEQUENCE

Interferon-γ (IFN-γ) binds with high affinity to heparan sulfate and heparin molecules through its carboxyl-terminal domain. In vivo, IFN-γ is eliminated from the bloodstream with a half-life (t1/2) of 1.1 min, due to binding to heparan sulfate. Unbound IFN-γ is cleaved rapidly at the carboxyl-terminal side, a process that removes at least 18 amino acids and inactivates the cytokine. When bound to heparin, the plasma clearance of IFN-γ is decreased greatly (t1/2 = 99 min), and the area under the curve obtained with IFN-γ alone represented only 15% of that obtained with injected IFN-γ bound to heparin. Furthermore, the binding of heparin to IFN-γ limits the extent of its carboxyl-terminal domain degradation to less than 10 amino acids. Importantly, this process increases the cytokine activity by as much as 600%. These data demonstrate that the blood clearance of the cytokine is a non-receptor-mediated process and that in vivo the local concentration of heparan sulfate/heparin-like molecules regulates IFN-γ activity by a unique mechanism involving a controlled processing of its carboxyl-terminal sequence.

Pancreatic Extracellular Matrix Alterations in Chronic Pancreatitis

The proliferation of pancreatic extracellular matrix, which characterizes chronic pancreatitis, has been analysed using immunohistochemistry. The relationship of matrix components to intraductal precipitates and the presence of serum proteins in precipitates were also studied to investigate the suggestion that ductal permeability increases in chronic pancreatitis. Pancreatic tissue from organ donors was compared with that from patients with chronic calcifying or chronic obstructive pancreatitis. Frozen sections were labeled with monospecific antibodies to collagen types I, III, pro-III and IV, laminin, fibronectin, IgG, IgA, and IgM and then visualized by indirect immunofluorescence. In chronic pancreatitis, interstitial collagens and fibronectin appeared increased and disorganized in both fibrous tissue and areas that appeared histologically normal. Type IV collagen distribution was abnormal and in some sites was present with interstitial collagen. In addition, intraductal precipitates were shown to contain immunoglobulins, and defects were identified in the duct basal lamina associated with precipitates. These results demonstrate that in chronic pancreatitis interstitial collagens are extensively disorganized, the fibrosis possibly being relatively labile. The presence of serum proteins in intraductal precipitates confirms an increase in ductal permeability, and associated defects in the basal lamina appear to define a route via which serum proteins may enter the intraluminal compartment.

Myofibroblastic stromal reaction in carcinoma of the breast: variations of collagenous matrix and structural glycoproteins

The matrix of mammary dysplasia, noninvasive ductal carcinoma, and invasive lobular and ductal carcinoma was analyzed by indirect immunofluorescence using antibodies to types I, proIII, III, and IV collagens, and laminin and fibronectin. Types proIII and III collagens were present in increased amounts in invasive carcinomas and were most abundant in the “young” edematous mesenchyme, areas corresponding to the peripheral invasive cellular front. Type I collagen was distributed throughout the matrix of invasive carcinomas but was most prominent within the central sclerotic zone of the neoplasms. Mammary dysplasia and noninvasive ductal carcinomas showed a uniform fibrillar and granular distribution of all types of collagen. In all but two cases of invasive carcinoma, staining with anti-laminin and anti-type IV collagen demonstrated the loss of basement membranes around tumor cells. In contrast, fluorescence pattern in noninvasive ductal carcinoma and dysplasia revealed an intact basement membrane. The distribution of fibronectin was similar to types proIII and III collagen. These findings support and extend our previous studies which suggested an analogy between the dynamics of matrix changes in granulation tissue and invasive carcinomas. These data also strengthen the concept that the myofibroblast could be a pivotal cell involved in the synthesis and redistribution of matricial proteins. The loss of basement membrane in invasive carcinomas appears to be an initial step for inducing the matricial alterations.

Immunofluorescent localization of collagen types I and III, and of fibronectin during feather morphogenesis in the chick embryo.

Abstract Collagen types I and III were purified from the skin of 3- or 7-week chickens and fibronectin from human serum. Antibodies were raised in rabbits and used in indirect immunofluorescence on frozen sections of 5- to 16-day chick embryo feather-forming skin. Prior to the formation of dense feather-forming dermis, anticollagen fluorescence was confined to a thin underlining of the dermal-epidermal junction (DEJ), while antifibronectin label was retained on loosely dispersed material in the predermal mesenchyme. Dense feather-forming dermis was characterized by loosening of the anti-collagen label along the DEJ, by its spreading throughout the thickness of dermis, and by an overall densification of antifibronectin label. Feather formation coincided with a decrease of anti-collagen and an increase of antifibronectin label density in the dermal feather condensations and in the core of outgrowing feather buds. By contrast, density of anti-collagen-labeled material was highest and anti-fibronectin-labeled material was lowest in interplumar and glabrous skin. In slanting feather buds and feather filaments, distribution of anti-collagen-labeled material exhibited a type-specific cranial-caudal asymmetry. The microheterogeneous distribution of extracellular matrix components might constitute part of the morphogenetic message that the dermis is known to transmit to the epidermis during the formation of cutaneous appendages.

Interferon-γ binds to heparan sulfate by a cluster of amino acids located in the C-terminal part of the molecule

Using three different approaches (domain mapping with monoclonal antibodies, limited enzymatic digestion and competition with synthetic peptides), we demonstrated that a cluster of basic amino acids on interferon‐γ is involved in its binding to heparan sulfate. This cluster (Lys131=Arg121) is localized in the C‐terminal part of IFN‐γ. Once bound to heparan sulfate, IFN‐γ is protected against protease attack.

Hepatic connective tissue changes in hepatosplenic schistosomiasis

Destruction of intrahepatic portal vein branches with dispersion of smooth muscle cells into the periportal fibrosis and preservation of arterial and ductal structures were the main characteristic findings seen in 66 surgical liver biopsies from patients with the hepatosplenic form of schistosomiasis. Besides these diagnostic features, the present histologic, immunocytochemical, and ultrastructural study revealed the presence of a complex matrix forming the portal and septal fibrosis in advanced schistosomiasis. There was marked hyperplasia of elastic tissue, presence of several collagen isotypes (I, III, procollagen III, IV, and V), actin, desmin, fibronectin, and laminin in a richly vascularized connective tissue. Signs of multifocal matrix (collagen) degradation were observed both at light and electron microscopic levels, suggesting a predominance of a fibrolytic process, at the time parasite-related lesions had almost disappeared. The latter findings are related to the involution of periportal fibrosis now being observed in patients who have undergone antischistosomal chemotherapy. They exemplify morphologic changes connected with chronic collagen degradation in human schistosomiasis that are similar to those first seen in experimental material. Evidence of either persistent or active chronic hepatitis was seen in several cases but its etiology could not be determined.

Perisinusoidal fibrosis and basement membrane-like material in the livers of diabetic patients

A direct correlation exists between collagenization of Disses space and the presence of diabetic microangiopathy in type I diabetes. To confirm and extend this finding, we studied four liver biopsy samples from two patients with type I diabetes (one with retinopathy) and two patients with type II diabetes (no retinopathy). All had normal or subnormal results on liver function tests and normal liver architecture. Levels of collagen types I, III, and IV, laminin, and fibronectin, as determined by immunocytochemical techniques, appeared increased in all patients. Liver biopsy samples were perfusion fixed for electron microscopy of sinusoids and sinusoidal cells. Numerous and thick collagen bundles could be seen in Disses space, as could the increase of basement membrane-like material underlying the endothelial cells, perisinusoidal cells, and sinusoidal membrane of hepatocytes. Perisinusoidal cells were active and had abundant rough endoplasmic reticula and thick processes. This preliminary study indicates that collagenization of Disses space is not specific to a certain type of diabetes. The increase of basement membrane-like material raises the question of whether liver sinusoids are truly different from other capillaries as far as diabetic microangiopathy is concerned.

Cell populations in the lesion of human cutaneous leishmaniasis: a light microscopical, immunohistochemical and ultrastructural study.

To characterize the in situ cellular immune response in localized cutaneous leishmaniasis (LCL), the authors studied frozen skin biopsies from 50 patients with LCL due toLeishmania braziliensis guyanensis. A panel of 31 monoclonal antibodies was used, which defined the number and distribution of inflammatory cell subsets. Skin inflammatory infiltrates were composed of T cells (with a local CD4/CD8 ratio of 1.05±0.7 vs 1.48±0.3 in peripheral blood), macrophages and a smaller number of B cells, natural killer cells and granulocytes. Most of the T cells expressed activation markers (interleukin-2 and transferrin receptors, HLA-DR+) and an increase in T-cell-receptorγδ expression was noted. Analysis of the CD4+ subpopulations with newly available reagents showed that helper T cells (CD4+CD45RO+) exceeded the suppressor/inducer subset (CD4+CD45RA+) by 1.4∶1. There were no differences between local immune variables from patients with primary infection (45 patients) and those with recurrence (5). In 7 patients, biopsies were analysed before and 1 month after specific treatment, and did not show significant differences except for a small increase of dermal CD1a+ (Langerhans) cells/mm2. The observed pattern of cellular skin infiltration suggests an immune-mediated tissue injury including T-cell-mediated cytotoxicity and delayed hypersensitivity reactions in addition to direct parasitic action.

Long-term administration of interferon-alpha in non-responder patients with chronic hepatitis C: follow-up of liver fibrosis over 5 years.

In chronic hepatitis C, previous data have shown that short‐term treatment with interferon‐α (IFN‐α) can reduce collagen deposition in the liver independently of the viral response. The aim of this work was to determine, in non‐responder patients, the long‐term effect of IFN‐α on liver fibrosis according to the total administered dose and the fibrotic stage. Fibrosis was investigated on liver biopsies from 24 non‐responder patients with chronic hepatitis C retreated with successive courses of IFN‐α. The degree of liver fibrosis was assessed on three successive biopsies, performed before IFN‐α treatment and 1 and 5 years later, in 13 and 11 patients, respectively, treated for less (mean: 7.5 months, 313 MU) and more (mean: 21.8 months, 791 MU) than 1 year. For each biopsy, fibrosis was assessed using a histological semiquantitative fibrosis scoring system and by morphometry after picrosirius red staining. Regardless of the dose and duration of IFN‐α therapy, a slight decrease of fibrosis was observed in patients 5 years after starting treatment. In cirrhotic patients, a short treatment induced an improvement followed by a relapse of fibrosis in 57%, and only 43% of patients showed constant collagen regression over the 5 years of follow‐up. On the contrary, after prolonged therapy, a progressive and significant decrease occurred throughout the follow‐up period in all patients (P = 0.045). Long‐term treatment with IFN‐α is therefore associated with regression of liver fibrosis, particularly in cirrhotic patients. These promising results need to be confirmed in a larger series of patients.

Fibronectin, Laminin, Type I, III and IV Collagens in Duchenne's Muscular Dystrophy, Congenital Muscular Dystrophies and Congenital Myopathies: An Immunocytochemical Study

The distribution pattern of fibronectin, laminin, type I, III and IV collagens in human skeletal muscle was studied by immunofluorescence. In normal muscle, as well as in congenital myopathies (CM), type I and III collagens were localized in the endomysium and the perimysium. Laminin and type IV collagen delineated precisely each muscle fiber but did not stain the perimysium. In Duchennes muscular dystrophy (DMD) as well as in congenital muscular dystrophies (CMD) the extensive proliferation of connective tissue consisted mainly of fibronectin and type I and III collagens. Laminin and type IV collagen delineated principally the basal lamina but suprisingly were found to be distributed to some extent all over the extracellular matrix. No disease--specific accumulation of components of the extracellular matrix was found which would enable us to differentiate these last two diseases, though the immunofluorescence reactions for all components were stronger in DMD than in CMD.