U. Di Tondo

Cell proliferation in the atherosclerotic plaques of cholesterol-fed rabbits Part 3. Histological and radioautographic observations on glucocorticoids-treated rabbits☆

Tritiated thymidine radioautography was employed to study the effect of cortisol and other glucocorticoids on cellular proliferation in the aorta and pulmonary artery of rabbits with cholesterol atherosclerosis. Labelled cell counts showed that glucocorticoids, even after one day and at a relatively low dose, decrease sharply the deoxyribonucleic acid synthesis in the intimal plaques. The hormonal influence on [3H]thymidine uptake seems to be a dose-dependent process. The relative potency of these steroids in inhibiting DNA synthesis in the plaques parallels closely their anti-inflammatory effectiveness. Conversely mineralocorticoids, including aldosterone and deoxycorticosterone, increase the rate of DNA synthesis in the plaques. It is concluded that the antiatherogenic effect of glucocorticoids on cholesterol-fed rabbits may be due, at least partly, to the inhibitory effect of these steroids on the DNA synthesis of the cellular components of the intimal plaques.

Calcium oxalate microdeposition in failing kidney grafts

Forty allograft nephrectomies (28 males and 12 females, mean age of 33.2 years), performed between 1986 and 1999, were selected. Twenty patients had been grafted from cadaveric donors; of the remaining cases, 15 had received a kidney from a living related donor. Except for six patients who received steroid-azathioprine immunosuppression, all recipients were treated with cyclosporine A and low doses of steroids. None had primary or acquired hyperoxaluria as the cause of original end-stage renal disease, and none had a history of kidney stones or urinary infection. Kidney failure occurred after a time period ranging from 2 days to 11 years posttransplant and was secondary to acute rejection in 17 cases, chronic rejection in 15, ischemic necrosis in five, acute or chronic pielonephritis in two, and allergic microangiopathy in one. No blood chemistry evaluations for oxalemia were performed. Clinical data are summarized in Table 1. Multiple sections cut from formalin-fixed, paraffin-embedded blocks and stained with haematoxylin-eosin (H&E) were examined using partially crossed polarizing screens. The presence of calcium oxalate crystals in different segments of the nephron or in the interstitium was recorded. Birefringent deposits with characteristic green iridescence, presenting as large plate-like or diamondshaped crystals, or any shape, provided that the crystals displaced tissue structures, were studied. Confirmation of the crystal nature was obtained using Yasue’s method, a histochemical stain considered to be specific for calcium oxalate, while usual stains for calcium (alizarin red and von Kossa’s method) were negative. Silver nitrate–rubeanic acid with 5% acetic acid pretreatment (Yasue’s method) stains only calcium oxalate, since calcium phosphate and calcium carbonate are dissolved with the acetic acid. A thyroid with multiple deposits of calcium oxalate and hydroxyapatite crystals was used as a positive control for the histochemical stains.

Ocular, cerebral and systemic interrelationships of cytomegalovirus infection in a postmortem study of AIDS patients

Purpose Eighty-six post-mortems of AIDS patients were reviewed microscopically and the presence of cytomegalovirus (CMV) infection in the viscera, brain and eye was recorded.Methods Immunohistochemical stains and in situ hybridisation with a CMV probe were performed.Results and conclusion CMV infection was observed in 63% of the cases. Visceral, cerebral and ocular involvement were overall 49%, 33% and 29%, respectively. The visceral form with no concomitant ocular and/or cerebral infection was the main cause of death (31%) in the 54 CMV-infected patients. Although CMV retinitis occurred mostly (20%) as a component of systemic disease, in 13% of the CMV-infected patients the eyes only were involved, while there were no cases with CMV limited to the brain. In the absence of systemic involvement, 9% of the cases showed concomitant ocular and cerebral infection, but because we failed to observe CMV optic neuritis without ocular involvement, retrograde viral spread from the brain through the optic nerve appears to be an infrequent mechanism of CMV retinitis.

Histological study of oxalosis in the eye and adnexa of AIDS patients.

Review of a series of 98 eyes removed at autopsy from 86 AIDS patients identified 12 cases (14%) showing varying degrees of microscopic calcium oxalate deposition. The oxalate crystals were birefringent using polarisation microscopy and were stained histochemically by the silver nitrate‐rubeanic acid method (Yasue), a stain considered to be specific for calcium oxalate. In two cases, the deposition was extensive and involved the surface of the ciliary processes, ciliary body and pars plana of the retina, the retinal and optic nerve blood vessel wall, a few retinal pigment cells, and the anterior inner sclera. A lesser degree of intraocular involvement was observed in the remaining 10 cases. In all but two eyes, where a peripheral active area of cytomegalovirus retinitis was present, no other significant microscopical abnormality was found. Clinically, these patients were asymptomatic. At autopsy, oxalate deposits were found in the kidney and/or thyroid in seven of the patients.

Steroids and Neuroendocrine Hormones Detected by the Immunoperoxidase Technique from Malignant Melanomas and Nevi of the Choroid and Conjunctiva

By the peroxidase-antiperoxidase technique, the authors studied 7 malignant choroidal melanomas, 7 conjunctival nevi and 1 malignant conjunctival melanoma with the aim to detect the presence of vasoactive intestinal polypeptide (VIP), adrenocorticotropic hormone (ACTH), gastrin, estradiol and testosterone. Positive staining reaction for VIP, estradiol and testosterone was observed in both malignant melanomas of the choroid and conjunctival nevi. The case of conjunctival melanoma was positive for VIP and ACTH but not for estradiol and gastrin.

Early mitochondrial calcifications in the rabbit aorta after adrenaline

Sequential fine structural changes were assessed in the aortic media of rabbits shortly after the intravenous injection of a massive single dose of adrenaline. Slight changes first appeared at 1 hour and by 6 hours marked changes were present which included accumulation of Alcian-stainable material in the intercellular spaces and alterations in mitochondria of smooth muscle cells. The mitochondria were swollen and showed decreased matrix density together with the presence of electron dense microcrystalline inclusions. Electron diffraction studies showed that these inclusions were composed by apatite-like crystals. The mechanism(s) involved and the possible pathological role of the mitochondrial calcification are discussed.

Metastasis of bronchial carcinoid tumour to choroid

A rare case of metastatic carcinoid tumour of the choroid is presented. Techniques used to identify the nature of the neoplasm (immunohistochemical or histochemical stains) and the numerous substances possibly secreted by the neoplastic cells (PAP immunocytochemical technique) are discussed.

Increased number of cancer cells in bronchial washing fluid detected by combining conventional cytology and high-resolution flow cytometry.

Abstract The present study was performed to improve early lung cancer diagnosis in bronchial washing fluid, thereby increasing the diagnostic sensitivity of bronchoscopy by means of high-resolution flow cytometry (FC). We combined dual-parameter DNA/protein FC and conventional cytology in bronchial washing fluid samples from 112 patients with neoplastic and non-neoplastic lung diseases and found 43% of histologically confirmed tumor cases to be cytologically positive; 63% of the tumor samples were aneuploid, 52% of the aneuploid cases were cytologically positive and 48% were negative. In the negative cases, FC was an independent diagnostic factor. In 32% of the cases, FC also failed to detect abnormalities. However, the combination of both techniques increased the sensitivity in detecting neoplastic cells to 73%. Furthermore, simultaneous DNA/protein analysis allowed the recognition of aneuploid cell lines not detectable by single DNA measurement. Identification of aneuploid subpopulations by dual-parameter analysis in cytologically negative one-parameter FC “diploid” samples assumes an important diagnostic value. Dual-parameter DNA/protein FC is a valuable technique that increases the diagnostic yield of bronchoscopy with no risk for the patient and a low additional cost.

44 SUCCESFUL ISOLATION, CULTURING AND DIFFERENTATION OF HEPATIC PROGENITOR CELLS (HPC) FROM HUMAN FOETAL LIVERS

Background and Aims: Vector-free approach developed by Gunther using PCR amplified full-length HBV genomes is one of the best in vitro system in reproducing the in vivo infection conditions. However, a major limitation of the method is the low level of intracellular HBV-DNA production. We investigated the reason(s) for this low efficiency. Methods: We analysed the replication fitness of HBV isolates from 11 untreated acute (3) or chronic (8) hepatitis B patients. Following Gunther’s method, the amplicons obtained by full-length PCR were cut by SapI to obtain linear HBV-DNA monomers competent for intracellular circularization upon transfection into HuH7 cells. Results: Seven (64%) of the 11 genome tested were replication competent in our system. Upon SapI cutting it was not possible by gel electrophoresis to distinguish digested from undigested HBVDNA, as both migrate with the same molecular weight. Direct sequencing of the region encompassing the catalytic site showed that in 19/20 experiments the majority of the HBV-DNA treated by SapI was undigested. Uncut HBV-DNA is considered unable to initiate replication because the Basic Core Promoter and Precore regions remains isolated. Since only a minority of the input DNA was replication competent, we hypothesized that this may be a reason for the low efficiency of the Gunther’s method. To overcome this limit we performed ligase treatment after SapI digestion obtaining the circularization of the monomer in vitro, then the fulllength circular-closed form was separated by gel electrophoresis for transfection. Densitometric quantification showed that in vitro circularization produced 4 times more replicative intermediates than the Gunther’s method. Furthermore, replicative intermediates were detected earlier in cells transfected with circular-closed monomers than with Gunther’s method (24 vs 48 hours). Conclusions: The low HBV replication efficiency of the Gunther’s method can be significantly improved by ligase treatment of the SapI digested full-length HBV-DNA prior transfection.