Shuttle of lentiviral vectors via transplanted cells in vivo.

Lentiviral vectors have turned out to be an efficient method for stable gene transfer in vitro and in vivo. Not only do fields of application include cell marking and tracing following transplantation in vivo, but also the stable delivery of biological active proteins for gene therapy. A variety of cells, however, need immediate transplantation after preparation, for example, to prevent cell death, differentiation or de-differentiation. Although these cells are usually washed several times following lentiviral transduction, there may be the risk of viral vector shuttle via transplanted cells resulting in undesired in vivo transduction of recipient cells. We investigated whether infectious lentiviral particles are transmitted via ex vivo lentivirally transduced cells. To this end, we explored potential viral shuttle via ex vivo lentivirally transduced cardiomyocytes in vitro and following transplantation into the brain and peripheral muscle. We demonstrate that, even after extensive washing, infectious viral vector particles can be detected in cell suspensions. Those lentiviral vector particles were able to transduce target cells in transwell experiments. Moreover, transmitted vector particles stably transduced resident cells of the recipient central nervous system and muscle in vivo. Our results of lentiviral vector shuttle via transduced cardiomyocytes are significant for both ex vivo gene therapy and for lentiviral cell tracing, in particular for investigation of stem cell differentiation in transplantation models and co-cultivation systems.

[Corneal dystrophies in the light of modern molecular genetic research]. / Hornhautdystrophien im Licht moderner molekulargenetischer Forschung.

Modern methods of molecular genetics have resulted in new insights into the pathogenesis of corneal dystrophies which now require a new classification. Clinical evidence, the results of histopathological and electron-microscopical examinations, especially in cases of recurrence which reflect early disease, and the immunohistochemical analysis of the deposits have already aroused the suspicion that the "old" classification dividing the dystrophies into those of the epithelium, of the so-called anterior membrane, of the stroma and of the endothelium may no longer be adequate. The detection of the BIGH 3 gene which is mainly expressed in the corneal epithelium, and its gene product keratoepithelin, have led to the insight that the so-called anterior membrane dystrophies (Reis-Bücklers, Thiel-Behnke) as well as the more common "classical" stromal dystrophies (granular dystrophies Types I and II, lattice dystrophies Types I and IIIA) are caused by different mutations of the above mentioned BIGH3 gene and are thus to be regarded as epithelial in origin. Lattice dystrophy Type II is part of the Meretoja syndrome, a systemic amyloidosis, and is caused by a mutation of the gelsoline gene on chromosome 9 (9q34). Gelsoline is also predominantly expressed in the corneal epithelium. In addition, the responsible genes, their gene-products and the mutations are known for Meesmann's epithelial dystrophy and for the so-called gelatinous drop-like dystrophy, while in other dystrophies only the location on a certain chromosome can be given, namely: 16q22 for the macular dystrophy, 1p36 for the central crystalline dystrophy of Schnyder and 20p11.2-q11.2 for the congenital hereditary endothelial and for Schlichting's posterior polymorphous dystrophies. As the production rate of new results in molecular genetics is very fast, the proposed new classification can only be of preliminary character.

Long term outcome of ocular adnexal lymphoma subtyped according to the REAL classification. Revised European and American Lymphoma.

AIM: To classify ocular adnexal lymphomas according to the Revised European and American Lymphoma (REAL) classification and to determine any correlation between clinical features or histomorphological variables with the patients' outcome. METHODS: Conventional and immunohistology were performed on representative sections of 53 specimens of 46 patients with ocular adnexal lymphoma. The antibodies used were CD20, BCL-2, CD21, CD23, CD43, CD3, CD5, p53, cyclin D1, pan-cytokeratin, kappa, lambda, IgD, and IgM. The growth fraction of the tumours was determined using the MIB-1 antibody directed against the Ki-67 antigen. Clinical follow up data regarding the outcome were obtained from the treating physicians and/or hospital files. The Student's t test and log rank test were used for statistical analysis. RESULTS: The patient collective consisted of 29 females and 17 males with an age range of 32-89.7 years (average 63 years). Almost all specimens represented B cell non-Hodgkin's lymphomas: extranodal marginal zone lymphoma (EMZL) (n=38), diffuse large cell B cell lymphoma (n=8), lymphoplasmocytic lymphoma/immunocytoma (n=2), mantle cell lymphoma (n=2), follicle centre lymphoma (n=1), and plasmacytoma (n=1). One case of a secondary anaplastic large cell lymphoma of T cell type (T-ALCL) was diagnosed. The majority of the patients had stage I disease. A variety of therapeutic regimens was administered, the main form of treatment being radiotherapy. The average follow up time was 85 months. Complete remission was achieved in 24 patients (10 after excision alone, eight after radiotherapy alone, three after combined excision and radiotherapy, one after chemotherapy alone, and two after combined radiotherapy and chemotherapy). 12 patients died of causes related to lymphoma; in one patient the cause of death was unknown. Six patients had persistent tumour at final follow up and two patients were lost to follow up. The stage at presentation, as well as the lymphoma malignancy category, had a significant correlation with the final course of the disease (p=0.0001 and p=0.03, respectively). A significant correlation was also noted between the final outcome (p<0.05) and tumour cell expression for Ki-67 antigen and p53 protein. CONCLUSION: 67% of patients with ocular adnexal lymphoma had EMZL. The stage at presentation had a significant influence on the final outcome. MIB-1 and p53 expression by the tumour cells proved to be important immunohistochemical markers concerning the prognosis. It is suggested that, following thorough staging investigations, primary EMZL (stage I) (if accessible) should be treated with excisional biopsy and subsequent low dose radiotherapy. Primary diffuse large cell B cell lymphoma of the ocular adnexa requires at least similar therapeutic measures and regular intensive follow up.

[Primary iris stromal cyst with unusual symptoms in an adult]. / Primäre Vorderkammerzyste mit ungewöhnlicher Symptomatik bei einem Erwachsenen.

BACKGROUND: Most epithelial cysts of the anterior chamber ("iris stromal cysts") occur after penetrating ocular injuries and represent secondary epithelial ingrowth. Primary iris stromal cysts are less common and mostly congenital. Acquired primary iris stromal cysts in adults are extremely rare and cause less often symptoms than congenital cysts. PATIENT: A 41-year old patient presented with sudden loss of visual acuity, epiphora and photophobia of his right eye. A large iris cyst was found in the nasal lower quadrant of the anterior chamber. It had not been present 3 years before when the patient was last seen by an ophthalmologist. There was no history of trauma and no signs of preceding ocular injury at slit-lamp examination. The cyst was surgically removed by iridocyclectomy. Postoperatively the patient developed cataract and macular edema. A phacoemulsification with posterior chamber lens implantation as well as a systemic treatment with steroids and acetazolamide were necessary. Until now, two years after surgery, the cyst did not recur. CONCLUSIONS: Primary iris stromal cysts also occur in adults. In contrast to previous reports the cyst of our patient has caused acute symptoms.

[Sequestrating senile scleral plaque simulating "necrotizing scleritis". Surgical management]. / Sequestrierender seniler Skleraplaque unter dem Bild einer "nekrotisierenden Skleritis". Operative Versorgung.

BACKGROUND: Presentation and surgical treatment of a sequestrating senile scleral plaque, a rare clinical disorder. PATIENT: A 76 year-old-female patient presented with foreign body sensation and epiphora. Half a year before, she had first noticed a "black spot" in her eye which had now progressed to a painless yellow-greyish plaque. CASE HISTORY: The patient had previously undergone a steroid-treatment elsewhere with a diagnosis "necrotizing scleritis" without any improvement. We found a yellow-greyish degenerative plaque sequestrating from the surrounding scleral tissue. In order to prevent superinfection and to secure the tissue defect we surgically removed the plaque and covered the remaining thin inner scleral tissue layers with autologous sclera and conjunctiva. The clinical diagnosis of a calcified senile scleral plaque was histologically confirmed. The patient has been free from any symptoms since the operation. To our knowledge this is the first description of surgical removal of a deeply sequestrating senile sceral plaque. CONCLUSION: Calcified sequesters represent a very rare complication of the otherwise frequent senile scleral plaques. Surgical intervention may be required to prevent superinfection and to cover the deep sceral defect. The clinical picture can be distinguished from necrotizing scleritis or scleromalacia perforans. Sequestrating senile scleraplaques do not tend to perforate. They are, however, a risk for infection and chronic inflammation.

[Orbital sarcoidosis--a case with an unusual course]. / Orbitale Sarkoidose--ein Fall mit ungewöhnlichem Verlauf.

BACKGROUND: Most cases of orbital sarcoidosis are associated with a systemic sarcoidosis. PATIENTS AND METHODS: A 67-year-old woman suffered from an orbital mass on the right side, which led to disturbance of the ocular motility. Slight improvement was achieved by the administration of systemic steroids. RESULTS: Histologically a chronic granulomatous inflammation was revealed in the biopsy of the orbital mass. The suspected diagnosis was sarcoidosis, but three conventional chest X-rays within 10 months and the serum angiotensin-converting-enzyme were normal. Suprisingly a computertomography of the chest showed mediastinal lymphomas. CONCLUSION: Granulomatous orbital inflammation without any local cause or other systemic granulomatous disease strongly suggests a systemic sarcoidosis. In case of missing lymph node enlargement in conventional chest X-ray computertomography should be performed.

Signet ring cell carcinoma of the eccrine sweat glands in the eyelid.

BACKGROUND: Signet ring cell carcinoma of the eyelid is a rare variant of eccrine sweat gland carcinoma and has been reported previously in only five patients. METHODS: The authors report the clinical findings of a 55-year-old man with a signet ring cell carcinoma in the left eyelid as well as a clinical follow-up of 4.5 years. Several biopsies and the exenteration specimen were analyzed by routine light microscopy, electron microscopy, and comprehensive immunohistochemical stains on paraffin sections. RESULTS: Histologically, the tumor was shown to be a rare type of eccrine sweat gland carcinoma with signet ring cells and Indian file growth pattern reminiscent of invasive lobular carcinoma of the breast. Estrogen and progesterone receptors were identified immunohistochemically. On electron microscopy, intracytoplasmic pseudolumina with microvilli were positive for anti-human milk fat globulin and the lectin peanut agglutinin. Clinically, the tumor followed a malignant course with orbital invasion and lymph node metastases. CONCLUSIONS: Histologic recognition of this variant of eccrine sweat gland carcinoma is important because of its aggressive and malignant behavior and the wide range of differential diagnoses. Primarily, metastatic mammary carcinoma must be excluded. The treatment is primary excision with histologic control of the excision margins. In more advanced stages, radiation therapy, neck dissection, and anti-estrogen therapy should be considered.

Malignant tumor of the retinal pigment epithelium with extraocular extension in a phthisical eye.

BACKGROUND: Malignant tumors of the retinal pigment epithelium (RPE) are exceedingly rare. We describe the histopathologic and immunohistochemical features of a RPE neoplasm that was found accidentally in a blind and painful phthisical eye. METHODS: The enucleated eye was investigated by light microscopy, and tumor tissue was also studied by electron microscopy. Immunohistochemistry was performed using antibodies against HMB-45, S-100 protein, NSE, cytokeratins, vimentin, desmin, GFAP, the HNK-1 carbohydrate epitope and alpha-smooth muscle actin. RESULTS: The tumor was located mainly in the vitreous cavity with practically complete destruction of the retina, but foci of choroidal infiltration and extraocular extension along vascular channels were identified. The mitotic rate was high, and large areas of necrosis were present. No features of differentiation were seen, apart from occasional desmosome-like junctions and deposition of basal lamina at the ultrastructural level. Adjacent to the tumor, reactive hyperplasia and metaplasia of RPE cells was prominent. By immunohistochemistry, tumor cells revealed intense immunoreactivity with anti-vimentin and weak staining with anti-S-100 protein. The hyperplastic RPE cells also reacted for cytokeratins 8, 18 and 19 and for alpha-smooth muscle actin. At more than 1 year post enucleation the patient is well and shows no signs of recurrence or metastatic disease. CONCLUSION: We present the features of a malignant tumor of the RPE with unequivocal extraocular extension. These findings raise the possibility that RPE hyperplasia may transform into a malignant tumor.

Vimentin and cytokeratin pattern in granular corneal dystrophy.

BACKGROUND: Corneal granular dystrophy is usually classified as a hereditary stromal disease of the cornea. Some investigations, however, have indicated an epithelial rather than a stromal origin of the granular deposits. In early stages and in recurrences of granular dystrophy after keratoplasty, the deposits are most often found in the upper microlayers of the cornea and even intraepithelially. METHODS: In this study we tried to identify immunohistochemical epithelial markers in the corneal granular deposits. RESULTS: A positive reaction with anti-cytokeratin 18 and polyclonal anti-vimentin were found both in the corneal epithelium and in the granular deposits. CONCLUSION: The immunohistochemical findings support the hypothesis of an epithelial origin of the corneal deposits in granular dystrophy.

Characteristic distribution of deposits in recurrent granular corneal dystrophy.

Granular corneal dystrophy can recur after penetrating keratoplasty. In those cases as in the early primary manifestation of the disease, deposits are found predominantly in the superficial corneal layers. At a later stage, stromal deposits can also be seen. We describe an additional particular location of deposits and correlate clinical with histological findings in three corneas from two patients. Deposits were found mainly superficially within the graft and around the edge of the transplant along the scar between host and donor cornea. We suggest that the deposits are of epithelial origin, though their production by stromal cells cannot be excluded. Chemical interactions are discussed as an explanation for the characteristic distribution of the deposits.

Sympathetic ophthalmia: an immunohistochemistry study of four cases.

We present histological and immunohistochemical data on four cases of sympathetic ophthalmia, a disease that is believed to occur predominantly after perforating injury to the eye. Only a few cases without previous perforation have been reported. Nevertheless, sympathetic ophthalmia should be taken into consideration if there is a bilateral intraocular inflammation, even without trauma, as in two of our cases (cases 1 and 2). An unusual case after uneventful intracapsular cataract extraction and a posttraumatic "classic" case are also presented (cases 3 and 4). We found a granulomatous infiltration of the uveal tract by lymphocytes, plasma cells, and epithelioid cells, particularly of the choroid. Dalen-Fuchs nodules were found in all cases, the second case also being associated with phacoanaphylaxis. Case 1 and 4 showed immunohistochemically a predominance of CD3-positive cells (T-lymphocytes), whereas in cases 2 and 3, many cells surprisingly stained positively for L26 (B-lymphocytes). In case 2 the immune response may have been altered by the additional phacoanaphylaxis. In all four cases, scattered epithelioid cells stained positively for CD 68. We conclude that in cases of bilateral uveitis, even without previous penetrating injury or after common intraocular surgery, sympathetic ophthalmia as a possible cause should be taken into consideration because an early diagnosis with subsequent enucleation of the exciting eye is of decisive influence on the course of the disease.

Immunoscintigraphy for ocular melanoma: a reliable diagnostic technique?

BACKGROUND: Immunoscintigraphy (IS) has recently been used as a diagnostic tool for ocular melanoma. We wanted to reevaluate published data in our own patients and to correlate immunoscintigraphic results with histologic findings and immunohistochemical characteristics of the tumour tissue. METHODS: During a 4-year period, IS was performed on 35 patients (average age 64 years) with suspected ocular melanoma by i.v. injection of 225.28S, a monoclonal antibody against high-molecular-weight melanoma-associated antigen. Histology was available in 22 cases. Tumour tissue was evaluated for cell type, vascularization, necrosis, pigmentation, and lymphocytic infiltration, and immunohistochemistry was performed with 225.28S and antibodies against HMB-45, S-100 and vimentin. One hundred and two patients with metastasizing cutaneous melanoma served as controls. In these patients the identical immunoscintigraphic technique was applied. RESULTS: IS yielded a positive result in about 50% of our patients with ocular melanoma, while in patients with cutaneous melanoma sensitivity was 89%. In five patients who turned out not to have melanoma, two false-positive results were obtained (one subretinal hemorrhage and one Wegener's granulomatosis). No correlation was found between any of the histological features or the immunoreactivity pattern and the immunoscintigraphic outcome. However, antigenic differences between ocular and cutaneous melanoma were evident. CONCLUSION: We conclude that IS, using the antibody applied in this study, is of only limited value in patients with ocular melanoma. Our results suggest that antigenic differences, rather than histological characteristics or technical problems, are responsible for the low sensitivity in ocular melanoma compared to cutaneous melanoma.