Immunohistochemical analysis of human orbital tissue in Graves' orbitopathy.

PURPOSE: Immunohistochemistry of orbital tissues offers a correlation between the microscopic changes and macroscopic clinical manifestation of Graves' orbitopathy (GO). Summarizing the participation of different molecules will help us to understand the pathogenesis of GO. METHODS: The pertinent and current literature on immunohistochemistry of human orbital tissue in GO was reviewed using the NCBI PubMed database. RESULTS: 33 articles comprising over 700 orbital tissue samples were included in this review. The earliest findings included the demonstration of HLA-DR and T cell (to a lesser extent B cell) markers in GO orbital tissues. Subsequent investigators further contributed by characterizing cellular infiltration, confirming the presence of HLA-DR and TSHR, as well as revealing the participation of cytokines, growth factors, adhesion molecules and miscellaneous substances. HLA-DR and TSHR are over-expressed in orbital tissues of GO patients. The inflammatory infiltration mainly comprises CD4 + T cells and macrophages. Cytokine profile suggests the importance of Th1 (especially in early active phase) and Th17 immunity in the pathogenesis of GO. Upregulation of proinflammatory/profibrotic cytokines, adhesion molecules and growth factors finally culminate in activation of orbital fibroblasts and perpetuation of orbital inflammation. The molecular status of selected parameters correlates with the clinical presentation of GO. CONCLUSION: Further investigation is warranted to define precisely the role of different molecules and ongoing search for new players yet to be discovered is also important. Unfolding the molecular mechanisms behind GO will hopefully provide insights into the development of novel therapeutic strategies and optimize our clinical management of the disease.

Atypical presentation of isolated orbital Langerhans cell histiocytosis.

BACKGROUND: A 9-year old female presented with one month of waxing and waning upper eyelid swelling. An excisional biopsy via anterior orbitotomy was performed. OBJECTIVE: To describe a patient presenting atypically with symptoms concerning for orbital cellulitis who was diagnosed with Langerhans cell histiocytosis (LCH). METHODS: Description of case report. RESULTS: We report a case of a 9-year old female with one month of periorbital edema and erythema suspected to be orbital cellulitis. A complete ophthalmological exam, subsequent imaging, and an excisional biopsy revealed the diagnosis of LCH. With a confirmed diagnosis, the patient started chemotherapy indicated by the Histiocyte Society Evaluation and Treatment Guidelines. CONCLUSION: Langerhans cell histiocytosis (LCH) embodies a spectrum of diseases with the primary pathologic process being the abnormal proliferation of polyclonal Langerhans cells. In children with isolated bony involvement, the most common presenting symptom is pain. Rarely is orbital involvement with associated periorbital edema and erythema the primary presentation.

Cribra orbitalia as a potential indicator of childhood stress: Evidence from paleopathology, stable C, N, and O isotopes, and trace element concentrations in children from a 17th-18th century cemetery in Jekabpils, Latvia.

Cribra orbitalia (CO), or porotic hyperostosis (PH) of the orbital roof, is one of the most common pathological conditions found in archaeological subadult skeletal remains. Reaching frequencies higher than 50% in many prehistoric samples, CO has been generally attributed to a variety of factors including malnutrition (e.g., megaloblastic anemia) and parasitism. In this study, we tested the relationship between CO, trace element concentrations, and stable isotope values (δ13C, δ15N, δ18O) in subadult skeletons from a 17th to 18th century cemetery in the historic town of Jekabpils, Latvia. A total of 28 subadults were examined, seven of which (25%) showed evidence of CO. Bioarchaeological evidence indicated high mortality for children in this cemetery: half of the burials were subadults under the age of 14, while a third were under the age of four. Life expectancy at birth was estimated to have been only 21.6 years. Trace element concentrations measured by Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) showed no relationship between presence or absence of CO and levels of manganese, zinc, strontium, barium, copper, cadmium, or lead in the bones (p>0.05). However, a significant correlation (p<0.05) was found between the presence of CO and decreased levels of iron. The correlations between CO and decreased levels of copper and lead approached significance (p=0.056 for both elements). Individuals with CO furthermore displayed significantly lower δ15N isotope values, suggesting greater consumption of lower trophic level food resources than those unaffected by CO; δ13C and δ18O values, in contrast, showed no significant differences. These results suggest that the prevalence of CO may be related to dietary deficiencies. In this case, low iron levels may also signify a diet low in other key vitamins (e.g., B9 and B12), which are known to cause megaloblastic anemia.

Conjunctival Melanoma With Morphologic Diversity and Orbital Invasion.

A 68-year-old male with a previous history of 3 dysplastic skin nevi was referred to ophthalmology for a 1-year history of a progressive growth of a firm nodule in the lower left eyelid. Examination revealed a firm nodule in the inferior anterior orbit and mild conjunctival pigmentation on the left inferior fornix. A conjunctival incisional biopsy was taken, showing a melanoma. Because of the infiltration by the mass of the orbit on magnetic resonance imaging, an exenteration was performed. Histopathological analysis showed a unique conjunctival melanoma showing morphological diversity; specifically, a pattern characteristic of conjunctival melanoma, and the concurrent presence of staghorn patterns, signet-ring cells, and rosettoid patterns. Seven years of follow-up shows that the patient is alive with no further metastasis or recurrence. This report represents the first documented case of multiple morphologic patterns within a conjunctival melanoma.

Proteomics of Orbital Tissue in Thyroid-Associated Orbitopathy.

CONTEXT: A potentially altered protein expression profile in orbital tissue from patients with thyroid-associated orbitopathy (TAO) is suspected. OBJECTIVE: To detect for the first time changes in proteomic patterns of orbital connective tissue in TAO and compare these with control tissue using mass spectrometry. DESIGN: Proteomics cross-sectional, comparative study. SETTING: Two academic endocrine institutions. SAMPLES: A total of 64 orbital and peripheral adipose tissue samples were collected from 39 patients with TAO and 25 control subjects. METHODS: Samples were analyzed and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technology. MAIN OUTCOME MEASURES: Mean intensity values of all identified peptides per protein. RESULTS: Thirty-one proteins were identified, of which 16 differentiated between controls and patients with TAO. Different protein patterns between orbital and peripheral adipose tissue were observed. Compared to controls, 10 proteins were markedly up-regulated (≥ 2-fold) in the orbital tissue of untreated patients: beta IV spectrin (6.2-fold), GTP binding G protein 2 (5.6-fold), POTE ankyrin domain family member F (5.4-fold), xylulokinase (4.1-fold), kinesin family member 1A and lipocalin 1 (both 3.6-fold), semicarbazide-sensitive metalloproteinase amine oxidase 3 and polymerase I transcript release factor (both 3.4-fold), cell-cycle protein elongin A binding protein 1 (3.3-fold), annexin A2 and cavin (both 3-fold), protein pointing to cell proliferation histone H4 (2.8-fold), and ADAM metallopeptidase with thrombospondin type 1 motif 14 (2.7-fold). The highest protein up-regulations were noted in the orbital tissue of medically untreated patients. Steroid therapy markedly reduced up-regulation of these proteins, foremost in nonsmokers. CONCLUSIONS: Proteins involved in tissue inflammation, adipose tissue differentiation, lipid metabolism, and tissue remodeling were up-regulated in orbital tissue of untreated patients with TAO. Steroids decreased the expression of these proteins, whereas smoking attenuated such effect.

Acoustic phonon-limited resistivity of spin-orbit coupled two-dimensional electron gas: the deformation potential and piezoelectric scattering.

We study the interaction between electron and acoustic phonons in a Rashba spin-orbit coupled two-dimensional electron gas using Boltzmann transport theory. Both the deformation potential and piezoelectric scattering mechanisms are considered in the Bloch-Grüneisen (BG) regime as well as in the equipartition (EP) regime. The effect of the Rashba spin-orbit interaction on the temperature dependence of the resistivity in the BG and EP regimes is discussed. We find that the effective exponent of the temperature dependence of the resistivity in the BG regime decreases due to spin-orbit coupling.

Interleukin-7 expression in tears and orbital tissues of patients with Graves' ophthalmopathy.

The aim of this study was to explore whether IL-7 participates in the pathogenesis of Graves' ophthalmopathy (GO). This was a prospective study. 20 GO patients (40 eyes) and 20 healthy volunteers (40 eyes) were recruited. The tear concentration of IL-7 was measured using ELISA assay. IL-7 expression in orbital tissues was evaluated by immunohistochemistry. Patients with inactive GO had the highest IL-7 concentrations in the tears, followed by healthy controls and patients with active GO per ELISA. Immunohistochemistry analysis showed that IL-7 expression in orbital tissues of the inactive GO samples was higher than that of the volunteers. Changes of IL-7 expression in different phases of GO suggested that IL-7 may play an important role in the pathogenesis of GO.

Kondo spin screening cloud in two-dimensional electron gas with spin-orbit couplings.

A spin-1/2 Anderson impurity in a semiconductor quantum well with Rashba and Dresselhaus spin-orbit couplings is studied by using a variational wavefunction method. The local magnetic moment is found to be quenched at low temperatures. The spin-spin correlations of the impurity and the conduction electron density show anisotropy in both spatial and spin spaces, which interpolates the Kondo spin screenings of a conventional metal and of a surface of three-dimensional topological insulators.

Masticatory loading, function, and plasticity: a microanatomical analysis of mammalian circumorbital soft-tissue structures.

In contrast to experimental evidence regarding the postorbital bar, postorbital septum, and browridge, there is exceedingly little evidence regarding the load-bearing nature of soft-tissue structures of the mammalian circumorbital region. This hinders our understanding of pronounced transformations during primate origins, in which euprimates evolved a postorbital bar from an ancestor with the primitive mammalian condition where only soft tissues spanned the lateral orbital margin between frontal bone and zygomatic arch. To address this significant gap, we investigated the postorbital microanatomy of rabbits subjected to long-term variation in diet-induced masticatory stresses. Rabbits exhibit a masticatory complex and feeding behaviors similar to primates, yet retain a more primitive mammalian circumorbital region. Three cohorts were obtained as weanlings and raised on different diets until adult. Following euthanasia, postorbital soft tissues were dissected away, fixed, and decalcified. These soft tissues were divided into inferior, intermediate, and superior units and then dehydrated, embedded, and sectioned. H&E staining was used to characterize overall architecture. Collagen orientation and complexity were evaluated via picrosirius-red staining. Safranin-O identified proteoglycan content with additional immunostaining performed to assess Type-II collagen expression. Surprisingly, the ligament along the lateral orbital wall was composed of elastic fibrocartilage. A more degraded organization of collagen fibers in this postorbital fibrocartilage is correlated with increased masticatory forces due to a more fracture-resistant diet. Furthermore, the lack of marked changes in the extracellular composition of the lateral orbital wall related to tissue viscoelasticity suggests it is unlikely that long-term exposure to elevated masticatory stresses underlies the development of a bony postorbital bar.

Lipid and fatty acid profile of the retina, retinal pigment epithelium/choroid, and the lacrimal gland, and associations with adipose tissue fatty acids in human subjects.

Accumulation of lipids within Bruch's membrane (BrM) and between BrM and retinal pigment epithelium (RPE) accounts for one of the biological changes associated with normal aging and may contribute to the development of age-related maculopathies. The origin of these lipids is still being actively investigated. The relative contribution of plasma lipids and lipids coming from the neural retina remains a matter of controversy. Low-density lipoproteins (LDLs) have been reported to significantly participate in the retina's lipid supply, after active remodeling within RPE. Meanwhile, RPE expresses the enzymatic machinery for synthesizing lipoprotein-like particles. The objective of this study was to establish associations between the fatty acid profile of the ocular structures and adipose tissue as a surrogate for the subjects' past dietary intake. Lipids and fatty acids were analyzed from the neural retina, retinal pigment epithelium (RPE)/choroid, the lacrimal gland, and adipose tissue, collected from 27 human donors (19 women, eight men) aged 59-95 years. DHA concentrations in the neural retina were positively associated with the concentrations in cholesteryl esters (CEs) from RPE/choroid and negatively associated with DHA concentrations in phospholipids (PLs) from RPE/choroid. DHA in orbital fat was positively associated with DHA in the lacrimal gland. No significant association was observed in the other ocular structures. Linoleic acid in orbital fat was positively associated with linoleic acid in the lacrimal gland, followed by the neural retina and CEs from RPE/choroid; it was slightly correlated with PLs from RPE/choroid. Other fatty acids that originate exclusively from the diet such as trans fatty acids were detected in orbital fat, the lacrimal gland, PLs, and CEs from RPE/choroid. DHA in the neural retina was poorly associated with its dietary intake, contrary to other fatty acids such as linoleic acid. Within this context, CEs may be important carriers of fatty acids entering the retina. Although epidemiological studies have reported the benefit of DHA in the prevention of age-related macular degeneration, the leading cause of blindness in Western countries, the relevance of supplementing patients with DHA is questioned.

[The inhibition effect of interleukin-1 receptor antagonist on interleukin-1 alpha-induced intercellular adhesion molecule-1 expression on orbital fibroblasts and adhesion of peripheral blood mononuclear cells].

OBJECTIVE: The aim was to identify whether interleukin-1 receptor antagonist (IL-1ra) inhibits interleukin-1 alpha (IL-1 alpha)-induced intercellular adhesion molecule-1 (ICAM-1) expression on cultured orbital fibroblasts, and adhesion of peripheral blood mononuclear cells (PBMC) to orbital fibroblasts, and to investigate the clinical application potential of IL-1ra in the treatment of Graves' ophthalmopathy (GO). METHODS: Cultured orbital fibroblasts from patients with GO and controls were challenged with IL-1 alpha or/and IL-1ra. Immunocytochemical staining was used to examine the changes of ICAM-1 in response to IL-1ra treatment; fluorescent photomicroscope was used to measure the adhesion between the labeled PBMC and orbital fibroblasts. Neutralizing antibody against ICAM-1 was used to demonstrate the role of ICAM-1 in the IL-1 alpha-induced adhesion. RESULTS: IL-1ra inhibits IL-1 alpha-induced ICAM-1 expression in cultured orbital fibroblasts both from GO patients and controls; IL-1ra inhibits IL-1 alpha-induced adhesion of PBMC to orbital fibroblasts in a concentration and time dependent manner. Moreover, a monoclonal anti-human ICAM-1 antibody produced a concentration dependent inhibition of the IL-1 alpha-induced adhesion of PBMC to the fibroblasts. CONCLUSIONS: IL-1ra inhibits IL-1 alpha-induced ICAM-1 expression in cultured orbital fibroblasts and the adhesion of PBMC to fibroblasts. IL-1 alpha-induced ICAM-1 expression may play an important role in the adhesion process. IL-1ra may be useful in the prevention or treatment of GO.

Effect of posterior temporal-parietal hematoma on orbital frontal chemistry in relation to a cognitive and anxiety state: a combined 1H-MRS and neuropsychological study of an unusual case as compared with 16 healthy subjects.

The authors report the unusual case of a 58-year-old woman (MJP) suffering from left temporal throbbing headache, associated with confusion. Magnetic resonance imaging showed a 5 x 3 x 2 cm hematoma at the left posterior temporal--parietal junction (PTPJ). Repeated MRI of MJP's brain performed during a 4-month follow-up period showed decrease in hematoma size (2.3 x 1.5 x 1) with evidence for development of encephalomalacia and resorption of blood products involving the area of hemorrhage. MJP had mild transcortical sensory aphasia characterized by difficulty with reading and processing, with semantic paraphasic errors while speaking and some difficulty with repetition. MJP had remained normotensive and seizure free, on Vasotec therapy and Dilantin prophylaxis. An in vivo proton magnetic resonance spectroscopy (1H-MRS) performed during an 8-month follow-up period showed reduced concentration for N-acetyl aspartate (NAA) by 19.3% (F=4.09, P<0.04), and myo-inositol by 32.0% (F=5.16, P<0.02) in the left orbital frontal cortex (OFC) as compared with 16 healthy subjects (age- and sex-matched). Cognitive tests (the Wechsler abbreviated scale of intelligence (WASI) and the Stroop color--word interference) showed a significant impairment suggesting involvement of higher-order cognitive functioning (memory, learning, and general intelligence) and attentional system. The Spielberger state-trait anxiety inventory (STAI) showed increased anxiety at the moment of the current examination and decreased tendency to be anxious over a long period of time. The Beck Anxiety and Depression Inventory revealed minimal anxiety and mild to moderate levels of depression. It is hypothesized that the PTPJ hematoma triggered long-distance pathways linking PTPJ area and frontal lobe, including OFC, which resulted in abnormal chemical changes in the left OFC and in cognitive tests impairment, and in long-term anxiety state changes.

The color difference in orbital fat.

OBJECTIVE: To identify and quantify carotenoids found in white and yellow orbital fat. METHODS: Specimens of nasal (white) and preaponeurotic (yellow) orbital fat were obtained from patients during upper eyelid blepharoplasty. Carotenoids and retinoids were extracted and subjected to spectral and high-performance liquid chromatography analyses. RESULTS: The chromophore content of extracts from unsaponified fat, as measured by absorbance at 425 nm per gram of fat, was 2- to 4-fold higher in preaponeurotic fat than in nasal fat. High-performance liquid chromatography analysis from enzymatically digested fat revealed large amounts of lutein, beta-carotene, and retinol and small amounts of other unidentified carotenoids. The amount of beta-carotene and lutein in preaponeurotic fat was approximately 4-fold higher than in nasal fat. CONCLUSIONS: The higher carotenoid content of preaponeurotic fat might cause it to be more yellow than other orbital fat, and lutein and beta-carotene might be selectively absorbed from plasma by preaponeurotic fat. CLINICAL RELEVANCE: The results provide baseline information for studies of the physiological features of orbital fat in normal and diseased conditions.

Characterization of human orbital fat and connective tissue.

This study was designed to evaluate the characteristics of human orbital fat and connective tissue. Two exenteration specimens were studied by light microscopy with special stains. Four distinct regions were identified on the basis of their connective tissue septa, which contained blood vessels and were composed of elastin and collagen types I, III, and IV. Transmission electron microscopy was performed on the opposite orbits. The fibroblasts and adipocytes appeared metabolically inactive and showed no regional differences. The fat was phase extracted from the connective tissue and subjected to biochemical analysis. No regional differences were found in the content of fatty acids and protein. The fatty acids included palmitic acid (22-24.6%), oleic acid (45-51.5%), and linoleic acid (15-18.6%). Despite demarcation of the orbital fat into distinct regions by the connective tissue septa, ultrastructural and biochemical analysis revealed no regional variations in the fat. The diagnostic and therapeutic implications of these findings are discussed.

Localization and clinical significance of thyrotropin receptor mRNA expression in orbital fat and eye muscle tissues from patients with thyroid-associated ophthalmopathy.

We have studied the cellular localization of thyrotropin receptor (TSH-R) mRNA in orbital fat and extraocular muscle tissues from patients with thyroid-associated ophthalmopathy (TAO) using Northern blot, reverse transcriptase polymerase chain reaction (RT-PCR), and in situ hybridization, and we correlated the findings with clinical estimates of ophthalmopathy. Although we failed to detect TSH-R mRNA in orbital tissues by Northern blot, TSH-R cDNA was amplified in orbital fat tissue from 13 of 25 patients with TAO and from 2 of 4 control subjects, in eye muscle tissue from 2 out of 7 patients with TAO, and in cultured orbital fibroblasts and subcutaneous fibroblasts from TAO patients. In situ hybridization showed that TSH-R mRNA was detected in cultured orbital fibroblasts as well as skin fibroblasts obtained from the patient. Furthermore, the expression of TSH-R mRNA in orbital fat tissue from patients with TAO significantly correlated with the orbital fat volume and the severity of ophthalmopathy, especially the extent of eye muscle dysfunction. These results suggest that the expression of TSH-R in the orbit, especially fibroblasts, may play a role in the pathogenesis and clinical manifestations of the ophthalmopathy in patients with TAO, although a secondary effect, involving fibroblasts in TAO is also possible.

TSH receptor in endocrine autoimmunity.

OBJECTIVE: The human thyrotropin receptor (hTSHR) is a potential common antigen in endocrine autoimmunity. Recently, some studies demonstrated transcripts for hTSHR or its components in the extrathyroidal tissue of patients with autoimmune thyroid disease (ATD), although others were unable to confirm these findings. In the present study we investigated orbital adipose/connective and muscle tissue as well as primary cell cultures of orbital fibroblasts and myoblasts from patients with thyroid and eye disease. METHODS: The messenger ribonucleic acid (mRNA) of hTSHR was reverse transcribed and amplified by polymerase chain reaction (PCR). To evaluate the existence of a functional hTSHR in cultured orbital fibroblasts and muscle cells, the TSH-mediated metabolic activity of the cells was measured by tetrazolium assay. RESULTS: We were unable to amplify the extracellular domain of hTSHR regardless of the material used. In contrast, transcripts of the transmembrane and intracellular domain of hTSHR were detectable in both crude retrobulbar tissue and primary cells cultures. The results of fibroblast amplification experiments were more successful than those with myoblasts. Furthermore, we were able to confirm that these transcripts of hTSHR can also be detected in the retro-ocular tissue of healthy persons. Independently of the TSH activity employed, no stimulation of fibroblasts or myoblasts was detected, even at higher TSH levels. CONCLUSION: These data do not suggest that hTSHR is expressed in a functional form in orbital tissue. However, a part of the receptor could play a role in the pathogenesis of autoimmune eye disease as a non-functional but antigenic protein. Whether a common antigen in the thyroid and orbit is related to hTSHR has not been clarified yet.

Interleukin-2 receptor beta and CD57 expression in orbital tissues from patients with chronic, stable thyroid-associated ophthalmopathy.

In order to determine whether components of the interleukin-2 receptor (IL-2R) and lymphoid cells are present in extra ocular and periocular tissues from patients with chronic, stable thyroid-associated ophthalmopathy (TAO) we studied 16 specimens of extra ocular muscle and periorbital connective tissue from 14 patients with chronic, stable, TAO using an immunohistochemical assay and a panel of murine monoclonal antibodies reactive with IL-2R alpha and beta components and lymphoid cell surface markers. As controls we studied orbital tissues from 11 patients undergoing surgery for unrelated orbital disorders. All extra ocular muscle specimens from patients with TAO exhibited IL-2R beta expression primarily on the perimysium and endomysium surrounding the ocular muscle fasciculi and fibers of which nine specimens stained intensely. The Natural Killer (NK) cell marker CD57 was the most common cell surface antigen detected, in seven of nine specimens, whose localization often corresponded to that of IL-2R beta distribution. No IL-2R alpha expression was detected in any specimen. Seven of the 11 control specimens were positive for IL-2R beta but in a less intense fashion than in TAO specimens while no CD57 staining was detected. T cell, B cell, and cells of granulocyte and monocyte lineage were only occasionally found in both TAO and control specimens. The aberrant expression of IL-2R beta and CD57 which may be representative of NK cell presence in extra ocular muscle tissues from patients with stable, chronic TAO may play a role in the pathogenesis of the ophthalmopathy.(ABSTRACT TRUNCATED AT 250 WORDS)

Profile of lambda light chain variable region genes in Graves' orbital tissue.

Graves' ophthalmopathy, a human autoimmune disease of unknown etiology, is strongly associated with autoimmune hyperthyroidism. A major controversy is whether retro-ocular muscle or orbital fat/connective tissue is the target of the immune response. Previously, we observed preferential PCR amplification of lambda (relative to kappa) light chain DNA from cDNA of Graves' orbital tissue-infiltrating B cells/plasma cells. There is little information on V lambda gene usage in man and none in diseased tissue. To characterize the orbital lambda light chains, we constructed cDNA libraries using PCR-amplified DNA from three tissues and sequenced the variable region genes from randomly selected clones. Analysis of 27 clones from orbital fat/connective tissue libraries from two patients with acute inflammatory eye disease, and 15 clones from orbital muscle of one of these patients, revealed a diverse spectrum of lambda V region genes. The nucleotide sequences of these 42 clones were most homologous to 12 different germline genes: four family I (subfamilies I-a, -b and -c), three family II, two family III and one family VII germline genes. Each orbital tissue had a distinct profile of V lambda sequences. However, all clones used J lambda 2/3 and all three orbital tissues contained clones related to family II genes. Although some clones had V region sequences in near germline conformation, the majority differed from the closest germline gene in both framework and complementarity determining regions. Whether or not these differences result from multiple germline gene usage or somatic mutation of a smaller number of germline genes cannot be determined until information on the V lambda repertoire and its polymorphisms is complete. However, the V lambda gene diversity we observed in both orbital muscle and orbital fat/connective tissue suggests a role for lambda autoantibodies in the pathogenesis of Graves' ophthalmopathy.

Clonality of benign lymphoid hyperplasia in orbit and conjunctiva.

In order to thoroughly characterize the clonal population of lymphoid hyperplasia of the orbit and conjunctiva, we investigated six cases which were histologically proven to be benign lymphoid hyperplasia. We analyzed the clonal rearrangements of the antigen receptors and bcl-2 gene, Epstein-Barr virus (EBV), and human T-cell leukemia virus type 1 (HTLV-I) by Southern blot and/or polymerase chain reaction (PCR), and performed in situ hybridization for mRNA of kappa and lambda immunoglobulin. Five cases showed rearrangements of immunoglobulin heavy chain gene (JH) and/or light chain gene (J kappa), and the monoclonal V-J recombination of JH in PCR. However, the rearranged bands were much more faint than was the germ-line band. We considered the monoclonal population of B cells small. Two of the five cases recurred locally after four and nine years respectively. Because benign lymphoid hyperplasias frequently contain an occult monoclonal B-cell population, a follow-up should be conducted. The remaining case in our investigation showed a rearrangement of the T-cell-receptor gene and proviral DNA of HTLV-I, and it showed rapid progress to adult T-cell leukemia after the biopsy. EBV and bcl-2 gene rearrangements were not observed in any of the six cases we studied.

Immunoglobulin A in Graves' orbital tissue: deoxyribonucleic acid amplification by polymerase chain reaction.

A role for IgA autoantibodies in Graves' ophthalmopathy is suggested by the presence of immunoglobulins of this class in Graves' orbital tissue, as detected by immunohistochemistry. We, therefore, investigated the possibility of using the polymerase chain reaction (PCR) to amplify IgA immunoglobulin genes from plasma cells infiltrating Graves' eye tissue. Template cDNA was reverse-transcribed from orbital muscle (M) mRNA of one patient (#7) and from orbital connective tissue/fat (F) mRNA of two patients (#1 and #7), both undergoing surgery for exophthalmos because of severe infiltrative ophthalmopathy. Preliminary studies to establish the PCR procedure were performed for kappa light chain DNA amplification. With the very small amount of orbital tissue template available, the sensitive "hot start" modification of the PCR was necessary to amplify significant amounts of kappa light chain DNA. Using this procedure, IgA heavy chain DNA was amplified from both connective tissue/fat (F7) and muscle (M7) cDNA of patient #7. The DNA yield was less for IgA than for IgG using the same template. There was no significant IgA (or IgG) DNA product using the connective tissue/fat cDNA of patient #1. While not implying that IgA-infiltrating plasma cells are specific for Graves' orbital tissue, our studies nevertheless demonstrate the feasibility of amplifying the genes coding for IgA antibodies from Graves' orbital tissue plasma cells. Expression of these immunoglobulin genes in future studies will make it possible to determine the antigen specificity of the antibodies expressed by Graves' orbital tissue plasma cells.