Clinicopathological correlation of choroidal neovascularization after external beam radiotherapy in age-related macular degeneration.

PURPOSE: To analyze the histopathology of choroidal neovascularization after external beam radiotherapy in age-related macular degeneration. METHODS: A retrospective non-case-matched comparative histopathologic study. The histoarchitecture of nine surgically removed subretinal specimens from nine patients that had undergone external beam radiotherapy for exudative age-related macular degeneration was studied. Seven patients had received 20 Gy in 10 fractions and two 15 Gy in 5 fractions with an average time interval between radiotherapy and surgical extraction of 14 months (range 3-28). A consecutive series of classic, mixed and occult choroidal neovascular membranes served as controls. RESULTS: Clinical findings. Radiation-associated choroidal neovasculopathy was angiographically suspected in four patients: a coarse net of vessels on fluorescein angiography developing at the border of previously irradiated choroidal neovascularization was observed in three patients; blebs at the margin of a plaque on indocyanine green angiography were observed in two patients. Pathological findings. Diffuse drusen as well as intra-Bruch's fibrovascular tissue was found in all irradiated specimens. In four specimens an edematous vascularized layer was seen between diffuse drusen and normal-appearing intra-Bruch's fibrovascular tissue. This lesion was not found in the control specimens. A particular correlation for the bleb lesion was not recognized. CONCLUSION: The appearance of an edematous subretinal pigment epithelial vascularized layer between diffuse drusen and normal-appearing fibrovascular tissue in four of nine irradiated membranes may be secondary to previous irradiation. It may correlate with the unusual exudative manifestations observed after external beam radiotherapy.

Clinicopathological correlation of retinal pigment epithelial tears in exudative age related macular degeneration: pretear, tear, and scarred tear.

AIMS: To analyse the histopathology of vascularised pigment epithelial detachments and tears of the retinal pigment epithelium (RPE) in age related macular degeneration (AMD). METHODS: The light microscopic architecture of 10 surgically removed subretinal specimens-three vascularised pigment epithelial detachments, four recent tears, and three scarred tears as a manifestation of AMD-were studied and correlated with the angiographic findings. RESULTS: Recent tears: a large fibrovascular membrane was found to be originally situated in Bruch's membrane. About half of the surface of the fibrovascular tissue was denuded of RPE and diffuse drusen. The RPE and diffuse drusen had retracted and rolled up, covering a neighbouring part of the intra-Bruch's fibrovascular membrane. The rolled up RPE and diffuse drusen were not interspersed with fibrovascular tissue but lay superficial to the intra-Bruch's fibrovascular membrane itself. Scarred tears: a collagen capsule surrounded the rolled up diffuse drusen and RPE. Fibrovascular tissue was found inside the rolled up material, predominantly at its choroidal side. CONCLUSION: The area of choroidal neovascularisation associated with a vascularised pigment epithelial detachment and a tear of the RPE may be larger than was hitherto thought or indicated by fluorescein angiography. This neovascular tissue may be present within the bed of the RPE tear, as well as at the site of the scrolled up RPE.

Clinicopathological correlation in exudative age-related macular degeneration: recurrent choroidal neovascularization.

PURPOSE: To report the pathology of surgically removed submacular tissue in recurrent choroidal neovascularization after laser photocoagulation of classic choroidal neovascularization in age-related macular degeneration. METHODS: A recurrent subfoveal choroidal neovascular membrane was surgically removed in two patients. The recurrence was identified as a classic membrane on fluorescein angiography at the foveal border of the laser scar. A net was visualized in the early venous phase of the indocyanine green angiogram, with associated late hyperfluorescence. Both patients had undergone laser photocoagulation for a classic interpapillomacular choroidal neovascular membrane about 1 1/2 years earlier. The specimens were serially sectioned and stained with hematoxylin-eosin, periodic acid-Schiff, Masson trichrome and phosphotungstic acid-hematoxylin. RESULTS: The two specimens consisted of subretinal fibrovascular tissue with fibrin exudation. Fibrovascular tissue bordered subretinal fibrous tissue adherent to Bruch's membrane and remnants of the choroid in one patient. The fibrovascular portion most likely corresponded to the recurrence, whereas the fibrous portion represented the original membrane, being obliterated after photocoagulation. Some peripapillary tissue was additionally removed in the other patient. The latter lesion was invisible on fluorescein angiography but stained in the late phase of indocyanine green angiography and corresponded histopathologically to poorly vascularized intra-Bruch's fibrovascular tissue. Granular deposits, periodic acid-Schiff positive and metachromatically purple on Masson trichrome stain, representing diffuse drusen (basal laminar/linear deposits), were identified in the three specimens. CONCLUSION: A subretinal fibrovascular membrane corresponded with the classic recurrent choroidal neovascularization.

Clinicopathological correlation of deep retinal vascular anomalous complex in age related macular degeneration.

AIMS: To analyse the histopathology of "deep retinal vascular anomalous complex" or "chorioretinal anastomosis". METHODS: Six patients with a deep retinal vascular anomalous complex (age range 66-88 years) had fundus photography and fluorescein angiography not more than 14 days before foveal translocation surgery. Four patients were also documented with indocyanine green angiography. The surgical specimens were serially sectioned and stained in a stepped fashion with Masson trichrome, periodic acid Schiff, and phosphotungstic acid haematoxylin, a histochemical stain for fibrin. RESULTS: A subretinal fibrovascular membrane was surrounded by a rim consisting of diffuse drusen (basal laminar deposits), retinal pigment epithelium, and amorphous, fibrinous material interspersed with remains of outer segments in all specimens. In two specimens vascular structures were identified that left the specimen towards the retina. Amorphous material with the remains of outer segments was not found on the retinal side of the fibrovascular tissue itself but in four specimens a small neuroretinal portion (outer nuclear layer) was adherent to the complex. In three specimens a thin fibrocellular membrane was seen at the choroidal side of the diffuse drusen. CONCLUSION: Deep retinal vascular anomalous complex represents histologically neovascularisation growing out of the neuroretina, into the subretinal space, which mimics choroidal neovascularisation. The term therefore appears rightly chosen.

Clinicopathological correlation in exudative age related macular degeneration: histological differentiation between classic and occult choroidal neovascularisation.

AIMS: To analyse the histopathology of classic and occult choroidal neovascular membrane surgical specimens in age related macular degeneration. METHODS: 35 membranes, from a consecutive series of surgically removed choroidal neovascular membranes in age related macular degeneration, were classified as classic or occult following the guidelines of the Macular Photocoagulation Study. Membranes with classic as well as occult components were considered as mixed membranes. The membranes were serially sectioned and stained with haematoxylin and eosin, Masson trichrome, periodic acid-Schiff, and phosphotungstic acid haematoxylin stain. The correlation has been made in a masked fashion. RESULTS: 31 membranes (19 classic, 10 occult, and two mixed membranes) could be analysed histologically. 18 classic choroidal neovascular membranes had a major subretinal fibrovascular component and 10 of these had an additional, minor fibrovascular component under the retinal pigment epithelium. The 10 occult membranes contained a fibrovascular component under the retinal pigment epithelium and the two mixed membranes contained fibrovascular tissue on both sides of the retinal pigment epithelium. Fibrin and remains of outer segments tended to occur at the lateral edges of classic membranes and to cover the inner surface of occult membranes. CONCLUSION: Classic choroidal neovascularisation in age related macular degeneration is predominantly composed of subretinal fibrovascular tissue while occult choroidal neovascularisation is composed of fibrovascular tissue at the choroidal side of the retinal pigment epithelium.

Different expression of the recombination activity gene RAG-1 in various populations of thymocytes, peripheral T cells and gut thymus-independent intraepithelial lymphocytes suggests two pathways of T cell receptor rearrangement.

The presence of transcripts of the recombination activating gene RAG-1 was studied by in situ hybridization on selected populations of murine thymocytes, peripheral lymphocytes and gut intraepithelial lymphocytes (IEL), obtained by cell sorting. RAG-1 mRNA was found in a majority of "double-positive" (DP) thymocytes, but was absent in "single-positive" thymocytes and peripheral T lymphocytes. The only other T lineages in which about 10%-20% of the cells contained RAG-1 mRNA, and in smaller amounts, were "double-negative" (DN), T cell receptor (TcR) gamma delta- cortical thymocytes and gut CD3- IEL. These observations suggest that (a) the high expression of RAG-1 transcripts in DP thymocytes is related to the process of expansion-selection of these cells, probably accompanied by repeated TcR rearrangements, and that (b) in contrast, CD3- IEL from the gut (which are thymus independent) as well as some DN thymocytes undergo limited TcR rearrangement giving rise locally to TcR+ T cells without prior extensive process of local expansion-selection. A small percentage of peripheral B cells also contained RAG-1 mRNA, raising the possibility that this protein may also be involved in immunoglobulin class switching.

Specific interferon genes are expressed in individual cells in the peritoneum and bone marrow of normal mice.

The use of a highly sensitive method of in situ hybridization capable of detecting one copy of IFN mRNA per cell showed that from 20-50% of the cells from the peritoneum and bone marrow of both normal pathogen-free and axenic mice exhibited grain counts significantly greater than background levels following hybridization with riboprobes specific for the mouse interferon-alpha (IFN-alpha), IFN-beta, or IFN-gamma genes. Labeling was shown to be specific, as the labeled probe was displaced by a 200-fold excess of the specific unlabeled probe but not by a 200-fold excess of an unrelated probe. Grain counts were reduced to background levels when cells were pretreated with ribonuclease prior to in situ hybridization. The extent of labeling with either IFN-alpha or IFN-beta-specific probes increased following i.v. inoculation of mice with the IFN-inducer Newcastle disease virus (NDV) whereas the degree of labeling observed with a probe specific for beta-actin remained unchanged. No significant differences were observed in the number of bone marrow or peritoneal cells that expressed IFN-alpha or IFN-beta mRNA from either high (C57B1/6) or low (BALB/c) IFN-producing strains of mice. The majority of IFN-alpha and IFN-beta-containing cells from both the bone marrow and peritoneum of normal pathogen-free and axenic mice resembled monocytes morphologically, whereas the majority of IFN-gamma mRNA-containing cells resembled small lymphocytes. In addition, in the bone marrow a number of large cells which resembled megacaryocytes were found to express high levels of IFN-alpha mRNA. Nuclear run-on assays showed that IFN-alpha and IFN-beta genes were actively transcribed in both bone marrow and peritoneal cells from normal and axenic mice. Low levels of de novo IFN-gamma RNA synthesis were detected in the nuclei of peritoneal cells only. The expression of IFN genes in individual cells in the tissues of normal animals may constitute a basis for the regulation of both homeostasis and host defense against virus infection and neoplastic cells.

Expression of the genes of class I interferons and interleukin-6 in individual cells.

The use of a highly sensitive method of in situ hybridization capable of detecting one copy of interferon (IFN) mRNA per cell coupled with quantitative analysis of cytokine mRNA showed that the number of copies of mRNA per cell was directly proportional to the logarithm of the number of silver grains formed over that cell. More than 90% of both virus-induced human Namalwa and mouse C243 cells exhibited grain counts significantly greater than background values following in situ hybridization with riboprobes complementary to human IFN- alpha and mouse IFN- beta mRNA, respectively. Labeling was shown to be specific, as the labeled probe was displaced by a 200-fold excess of the specific unlabeled probe but not by a 200-fold excess of an unrelated probe. Although the large majority of cells within a population responded to induction, considerable variation was observed, however, in the content of IFN mRNA per cell: 24% of induced C243 cells contained more than 50 copies of IFN-beta mRNA per cell while 60% of the cells contained 10 copies or less. Low levels of IFN mRNA were also detected in both uninduced C243 cells and uninduced Namalwa cells. Five to 10% of peripheral blood mononuclear cells from normal donors expressed INF-alpha mRNA following induction in vitro. Approximately 1% of untreated peripheral blood mononuclear cells also exhibited low levels of IFN-alpha mRNA. Analysis of interleukin-6 (IL-6) mRNA showed that 97% of TNF-induced human MG63 cells contained IL-6 mRNA, although, again, the amount varied considerably from cell to cell.

Genes for interleukin-1, interleukin-6, and tumor necrosis factor are expressed at markedly reduced levels in the livers of patients with severe liver disease.

The genes for interferon (IFN) alpha, IFN gamma, IL-1 beta, IL-6, and TNF alpha were transcribed at readily detectable levels both in liver biopsies from individuals with normal liver function and in samples of normal viable liver taken for transplantation. These results provided evidence for the concept that such multifunctional cytokines play a role in homeostasis in normal human tissues. In normal human liver, in situ hybridization studies showed that, in the absence of a detectable inflammatory response, both hepatocytes and mononuclear cells exhibited a similar degree of expression of IL-6 mRNA in keeping with the finding that IL-6 is produced by cells of different lineages. The levels of IL-1, IL-6, and TNF mRNA were found to be markedly reduced in extracts of the livers of patients with primary biliary cirrhosis and other forms of autoimmune liver disease at a time when extensive liver lesions were apparent, compared to the levels of expression of these cytokines in the livers of normal individuals. The reduced expression of IL-1, IL-6, and TNF mRNAs appeared to be a specific effect and not due to a general reduction in RNA synthesis as the IFN alpha, IFN gamma and actin mRNAs were expressed at similar levels in both normal and diseased livers. The levels of IL-1 beta, IL-6, and TNF mRNAs were also reduced in samples of liver from a patient with a drug induced fulminant hepatitis suggesting that this specific pattern of altered cytokine gene expression was characteristic of the advanced stage of severe liver disease.

Protection of cattle from infection with vaccinia virus by bovine interferon alpha C.

The efficacy of bovine interferon alpha C as an antiviral compound was evaluated in calves infected with vaccinia virus. Calves treated with the interferon did not develop characteristic vaccinia lesions, demonstrating a clear protective effect of treatment. The only side effect observed was hyperthermia. After intramuscular injection, interferon appeared quickly in the blood and was cleared within 24 hours. The in vivo antiviral activity of bovine interferon alpha C was confirmed by its ability to induce 2'5' A synthetase, an enzyme implicated in the antiviral state, in lymphocytes.

Characterization of interferon-alpha binding sites on human cell lines.

The binding sites for human interferon-alpha (IFN-alpha) have been characterized on human lymphoblastoid, melanoma, rhabdomyosarcoma, and cervical carcinoma cells. Crosslinking of iodinated-recombinant DNA-derived IFN-alpha-Con1, an analog of the known IFN-alpha subtypes, to the cell surface with disuccinimidyl suberate yielded four IFN-receptor complexes of 118, 138, 159, and 260 kD on all cell lines that specifically bind IFN-alpha. Since IFN-alpha exists in solution as monomers, dimers, and trimers, and the three lower molecular weight IFN-alpha-receptor complexes differ by the molecular weight of IFN-alpha (20 kD), this suggests that the human IFN-alpha receptor of 100 kD binds more than one molecule of IFN-alpha. The higher molecular weight complex of 260 kD may result from dimerization of the receptor. None of these complexes was observed in a rhabdomyosarcoma subclone that does not specifically bind IFN-alpha. Pretreatment of cells with trypsin abolished the formation of these complexes. Pretreatment of cells with neuraminidase did not reduce IFN-alpha binding, but increased the electrophoretic mobility of all four IFN-alpha-receptor complexes. Other glycosidases (i.e., mannosidase, beta-galactosidase, and endoglycosidase F) had no effects on IFN-alpha binding or mobility of complexes. Thus, although the IFN-alpha receptor is a glycoprotein, the glycosylated portion is apparently not part of the IFN-alpha-binding domain. The formation of IFN-alpha-receptor complexes is independent of the duration of incubation with IFN (from 5 min to 1 h at 15 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)

Antiviral effect of bacterially produced human interferon (Hu-IFN alpha 2) against experimental vaccinia infection in calves.

The heterologous antiviral efficiency of bacterially produced human interferon (Hu-IFN alpha 2) in the bovine species was studied, using vaccinia infection as experimental model. In a double blind experiment, young calves were intramuscularly injected daily for seven consecutive days with different doses of Hu-IFN alpha 2 or placebo, the treatment starting 24 h before intradermal inoculation of vaccinia virus. A clear protection by interferon was observed in all the IFN treated animals, although individual variations in the sensitivity to IFN were recorded. The efficiency of treatment varied according to the dose of IFN used: With the highest dose (10(6) IU/kg), complete protection could be obtained. The only side-effect observed was hyperthermia. Circulating antiviral activity appeared quite early after each IFN injection, presented a more or less biphasic kinetics, and was completely cleared after 24 h, justifying the daily treatment schedule. The first evidence of an in vivo antiviral effect of human interferon in the bovine species opens broad perspectives for a future use of interferon in veterinary medicine.

Experimental rotavirus diarrhoea in colostrum-deprived newborn calves: assay of treatment by administration of bacterially produced human interferon (Hu-IFN alpha 2).

Seven colostrum-deprived newborn calves were orally inoculated within 24 hours after birth with bovine rotavirus. Three of them were intramuscularly injected with bacterially produced human interferon (Hu-IFN alpha 2). The four control animals presented a severe diarrhoea for at least 48 hours, while only one of the treated calves suffered from a transient diarrhoea for a few hours. Hu-IFN alpha 2 seems therefore able to control rotavirus diarrhoea in newborn calves, although it did not inhibit virus excretion and seroconversion in the treated animals. Moreover, the administration of endogenous interferon appeared to be well tolerated by newborn calves. The efficacy of human alpha 2 interferon for the treatment of this important virus infection of cattle seems thus well established.

Interferon response in colostrum-deprived newborn calves infected with bovine rotavirus: its possible role in the control of the pathogenicity.

Colostrum-deprived newborn calves were experimentally infected with cell-culture rotavirus. A similar process of infection was observed when the animals were inoculated immediately after birth or at the age of three days, with a corresponding delay in the onset of virus excretion and interferon production in the later case. With high doses of virus, interferon was produced very early and no symptoms were observed. With lower doses of virus, interferon production was delayed and preceded by a severe but transient diarrhoea. In all cases, several waves of interferon production were observed. Our data indicate that interferon plays an important role in the control of viral diseases in calves and in their natural recovery.

[Attempt at the experimental reproduction of rotavirus diarrhea in newborn calves deprived of colostrum]. / Tentative de reproduction expérimentale de la diarrhée à rotavirus chez des veaux nouveau-nés privés de colostrum.

Colostrum-deprived newborn calves were orally inoculated with different doses of cell-culture bovine rotavirus or with bacterium-free filtrates of calf stools containing rotavirus. None of the animals that received high doses of cell-culture rotavirus developed diarrhoea or any other clinical sign, although all of them excreted virus for several days and produced specific antibodies; calves inoculated with lower doses of cell-culture virus or with stool filtrates showed a transient diarrhoea 48 h after inoculation. Such paradoxical results might be due to a phenomenon of interference, as bovine rotavirus is susceptible to interferon. In experimental conditions, rotavirus produces only a mild and transient diarrhoea: this contrasts with the situation observed in farms, where that virus may provoke important problems. In association with the virus itself, numerous other factors such as the environmental conditions or the response of the calf to the infection also play a role in the evolution of the disease.