Multivariate modelling of prostate cancer combining magnetic resonance derived T2, diffusion, dynamic contrast-enhanced and spectroscopic parameters.

OBJECTIVES: The objectives are determine the optimal combination of MR parameters for discriminating tumour within the prostate using linear discriminant analysis (LDA) and to compare model accuracy with that of an experienced radiologist. METHODS: Multiparameter MRIs in 24 patients before prostatectomy were acquired. Tumour outlines from whole-mount histology, T2-defined peripheral zone (PZ), and central gland (CG) were superimposed onto slice-matched parametric maps. T2, Apparent Diffusion Coefficient, initial area under the gadolinium curve, vascular parameters (K(trans),Kep,Ve), and (choline+polyamines+creatine)/citrate were compared between tumour and non-tumour tissues. Receiver operating characteristic (ROC) curves determined sensitivity and specificity at spectroscopic voxel resolution and per lesion, and LDA determined the optimal multiparametric model for identifying tumours. Accuracy was compared with an expert observer. RESULTS: Tumours were significantly different from PZ and CG for all parameters (all p < 0.001). Area under the ROC curve for discriminating tumour from non-tumour was significantly greater (p < 0.001) for the multiparametric model than for individual parameters; at 90 % specificity, sensitivity was 41 % (MRSI voxel resolution) and 59 % per lesion. At this specificity, an expert observer achieved 28 % and 49 % sensitivity, respectively. CONCLUSION: The model was more accurate when parameters from all techniques were included and performed better than an expert observer evaluating these data. KEY POINTS: • The combined model increases diagnostic accuracy in prostate cancer compared with individual parameters • The optimal combined model includes parameters from diffusion, spectroscopy, perfusion, and anatominal MRI • The computed model improves tumour detection compared to an expert viewing parametric maps.

Estrogen receptors and biologic response in rat parathyroid tissue and C cells.

The expression of the PTH and calcitonin genes is dramatically decreased by 1,25(OH)2D3 in vivo, and the PTH gene expression is increased by hypocalcemia. We have now studied the effect of estrogens on the expression of these genes in vivo. 17 beta-Estradiol, given to ovariectomized rats, led to a fourfold increase in PTH mRNA and calcitonin mRNA levels. These effects occurred 24 h after single injections of 37-145 nmol estradiol, or after constant infusions of 12 pmol/d for 1 or 2 wk, where there was no effect on serum calcium levels. The estrogen receptor mRNA was demonstrated in the thyroparathyroid tissue by polymerase chain reaction. The estrogen binding was localized to the parathyroid and C cells by immunohistochemistry. Uterus weight was increased by repeated larger doses (73 nmol/d x 7) of estradiol, but not by the small doses (12 pmol/d for 1 or 2 wk) which were effective on the PTH and calcitonin genes, suggesting a sensitive endocrine effect. These results confirm that the parathyroid and C cells are target organs for estrogen, leading to an increased expression of PTH and calcitonin, which by their combined anabolic effect on bone would help prevent osteoporosis.

Parathyroid cell proliferation in normal and chronic renal failure rats. The effects of calcium, phosphate, and vitamin D.

Secondary hyperparathyroidism is characterized by an increase in parathyroid (PT) cell number, and parathyroid hormone (PTH) synthesis and secretion. It is still unknown as to what stimuli regulate PT cell proliferation and how they do this. We have studied rats with dietary-induced secondary hyper- and hypoparathyroidism, rats given 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and rats after 5/6 nephrectomy for the presence of PT cell proliferation and apoptosis. PT cell proliferation has been measured by staining for proliferating cell nuclear antigen (PCNA) and apoptosis by in situ detection of nuclear DNA fragmentation and correlated with serum biochemistry and PTH mRNA levels. A low calcium diet led to increased levels of PTH mRNA and a 10-fold increase in PT cell proliferation. A low phosphate diet led to decreased levels of PTH mRNA and the complete absence of PT cell proliferation. 1,25 (OH)2D3 (25 pmol/d x 3) led to a decrease in PTH mRNA levels and unlike the hypophosphatemic rats there was no decrease in cell proliferation. There were no cells undergoing apoptosis in any of the experimental conditions. The secondary hyperparathyroidism of 5/6 nephrectomized rats was characterized by an increase in PTH mRNA levels and PT cell proliferation which were both markedly decreased by a low phosphate diet. The number of PCNA positive cells was increased by a high phosphate diet. Therefore hypocalcemia, hyperphosphatemia and uremia lead to PT cell proliferation, and hypophosphatemia completely abolishes this effect. Injected 1,25 (OH)2D3 had no effect. These findings emphasize the importance of a normal phosphate and calcium in the prevention of PT cell hyperplasia.

Induction of lipid storage in cultured leukemic myeloid cells by pyrene-dodecanoic acid.

When incubated for 1-3 days in the presence of the fatty acid analog, 12-(1-pyrene)dodecanoic acid, the neutral lipid content of cultured human leukemic myeloid cells increased considerably, while that of the phospholipids increased to a much lesser extent. Among the neutral lipids, di- and monoacylglycerols predominated and a considerable portion of the fatty acyl residues of these newly synthesized neutral lipids consisted of pyrene-dodecanoic acid. Light microscopy showed evidence for the presence of highly fluorescent lipid droplets within the cells. Electron microscopy showed lipid globules, mostly devoid of a unit membrane, multivesicular inclusion bodies and some multilamellar membranous structures. In comparison, cells incubated with palmitic acid show neither these cellular structures, nor the increase of the neutral lipid content. The lipid storage, induced by pyrene-dodecanoic acid, is probably related to ineffective degradation of this fatty acid analog and might serve as an experimental model of cellular lipidosis.

Biochemical and ultrastructural studies on an Epstein-Barr virus-transformed lymphoid cell line from a Niemann-Pick disease type C patient.

Human lymphoid cell lines established from normal subjects and from a Niemann-Pick disease type C patient were investigated from a triple point of view of enzymology, metabolism and ultrastructure: Sphingomyelinase activities, isoenzyme electrofocusing profiles and properties of the major enzyme were quite similar in type C and normal lymphoid cell lines. Similarly, no significant difference was observed in non-specific phosphodiesterases hydrolysing bis(methylumbelliferyl)phosphate and bis(methylumbelliferyl)pyrophosphate. The study of the lipid composition of type C cells showed no obvious accumulation of sphingomyelin or other phospholipid, but only a higher amount of glycolipids (mainly GlcCer and GbOse3Cer), as visualized by bidimensional thin-layer chromatography. Ultrastructural studies demonstrated, in type C cells, the presence of an obvious lysosomal storage of amphiphilic lipids quite similar to that observed in tissues of type C patients. These studies, which demonstrate the validity of lymphoid cell lines as an experimental model system for type C disease, agree with the current opinion that an impairment of sphingomyelin catabolism is not the primary defect in type C disease.

Intra-operative assessment of excision margins using breast imprint and scrape cytology.

Local recurrence in breast cancer surgery is related to the completeness of excision. Histological analysis of excision margins is time consuming and impractical for use intra-operatively. Our group evaluated breast imprint and scrape cytology (ISC) for the assessment of excision margins in a feasibility study in 1993-4, with 10 year clinical follow-up. Twenty-six consecutive women undergoing 27 wide local excisions for breast cancer had excision margins prospectively assessed with intra-operative ISC blinded to histology. All ISC results were ready (range 22-30 min) before surgery was completed. ISC agreed with histology in 21/27 (=78%) and disagreed in 6/27 (=22%) of the cases. In two cases with local recurrence, histology was positive in one case, whereas ISC margins were positive in both. Intra-operative ISC is reliable and could help the surgeon to excise more tissue to prevent a second (re-excision) operation. ISC margins may predict clinical outcome, although a larger interventional follow-up study is required.

Experimental vascular occlusion in hypercholesterolemic rabbits.

Rabbits fed a diet rich in cholesterol developed hypercholesterolemia and atheromas in the cornea, sclera, iris, ciliary body, and choroid, and a few synchysis scintillans in the vitreous. The fellow eye of these rabbits, in which one nasal vortex vein was occluded, developed far more severe changes in all the above-mentioned eye tissues, especially over the sector of the eye drained by the occluded vein. In hypercholesterolemic rabbits in which a short posterior ciliary artery was occluded, the ischemic area of the choroid was gradually seem to become infiltrated with large crystalline particles. The combination of abnormal blood flow and hypercholesterolemia gives rise to an exaggerated deposition of extravascular lipid material. This experimental model is probably relevant to such human conditions as drusen and lipoid deposition in the choroid and cornea and the formation of asteroid hyalosis.

Systemic lupus erythematosus associated with acute Epstein-Barr virus infection.

Systemic lupus erythematosus (SLE) is a multisystem disease of unknown origin, characterized by a variety of autoimmune phenomena. Viruses have long been postulated to play a role in its pathogenesis. Several observations suggested a link between Epstein-Barr virus (EBV) and SLE. We describe a 14-year-old girl who presented with acute onset of SLE concurrently with clinical and laboratory findings consistent with EBV-induced infectious mononucleosis (IM). Evidence for acute EBV infection was confirmed by serological studies and detection of specific EBV antigens on kidney biopsy. This close association between EBV and SLE suggests a possible role of the virus in the pathogenesis of SLE in this patient.

Clonal analysis of three morphologically distinct lymphomas occurring in the same patient.

AIMS: To determine whether three morphologically distinct lymphomas (mucosa associated lymphoid tissue (MALT) lymphoma, monocytoid B cell lymphoma, and large cell anaplastic lymphoma), which occurred in the same patient, were in fact three morphological variants of the same lymphoproliferative process. METHODS: Previously described methods of clonal analysis using the polymerase chain reaction (PCR) were used to determine the pattern of rearrangements of the immunoglobulin heavy chain gene and the T cell receptor beta and T cell receptor gamma chain genes in the three lymphomas. RESULTS: All three morphological entities had identical patterns of gene rearrangements. CONCLUSIONS: This finding confirms the association between MALT lymphoma and monocytoid B cell lymphoma, and also provides evidence that large cell anaplastic lymphoma may not only arise de novo but may also be an end stage morphological picture in lymphoma progression.

Rapid isolation and identification of reovirus-3.

Mammalian reoviruses are connected with a variety of humans diseases, including gastroenteritis, malabsorption and hepatitis. Recently, reovirus-3 was found to be associated with neonatal biliary atresia. We describe a technique for the rapid isolation and identification of reovirus-3. Mouse fibroblasts (L 929 cells) were grown in monolayers in a RPMI 1640 medium containing 10% calf serum. The cytopathic effects were visualized by the rounding of the L 929 cells and the appearance of fine granulation in the cytoplasm 48 h after the infection. Hematoxylin-eosin staining showed swelling and rounding of the infected cells, diminished chromatin in the nuclei, and the absence of mitoses. The immunohistochemical staining by the avidin-biotin-peroxidase technique was positive in the infected monolayers of the L 929 cells. The positive staining was limited to cytoplasmic inclusions, which were surrounded by a halo and sometimes by vacuoles. We conclude that the described technique is useful for the rapid isolation and identification of reovirus-3.

Conjunctival ultrastructure in Niemann-Pick disease type C.

A 5-year-old girl with Niemann-Pick disease type C had normal eyes but the conjunctival ultrastructure was abnormal. Lamellar cytoplasmic bodies, characteristic of Niemann-Pick disease, were found in epithelial cells, stromal fibroblasts, endothelial cells, and pericytes of the stromal capillaries. The basement membrane of the capillaries was multilayered. The Golgi apparatus was unusually well developed. The combination of ocular findings and conjunctival ultrastructure may be helpful in the differential diagnosis of Niemann-Pick disease and its subtypes.

MIB-1 and PC-10 immunostaining for the assessment of proliferative activity in primary acquired melanosis without and with atypia.

AIMS: To compare the proliferative activity of intraepithelial melanocytes in primary acquired melanosis (PAM) without atypia and PAM with atypia by immunohistochemical staining for the Ki-67 antigen and the proliferating cell nuclear antigen (PCNA). METHODS: Formalin fixed, paraffin embedded sections from 35 archival specimens of PAM without atypia (n = 19) and with atypia (n = 16) were studied by immunostaining with MIB-1 and PC-10 monoclonal antibodies that react with the Ki-67 antigen and PCNA respectively. The results were calculated as the mean number of positive cells per eyepiece grid. All specimens were evaluated by two masked observers, and the interobserver reproducibility was assessed. RESULTS: The means of the positive cell count in PAM with atypia were significantly higher compared with PAM without atypia for both observers, in both the PC-10 and the MIB-1 stained sections. In a linear least square model that estimated the interobserver and between group variation, the difference of MIB-1 and PC-10 positive cell count between PAM without and with atypia remained highly significant. The difference between the observers was not significant. CONCLUSIONS: Immunostaining with MIB-1 and PC-10 demonstrated that PAM with atypia has higher proliferative activity than PAM without atypia. This method was found to be reproducible between different observers.

Retrospective confirmation of Mycoplasma infection by the immunoperoxidase technique.

Rat tissues embedded in paraffin blocks stored for 8-10 yr were sectioned for immunoperoxidase staining. Sections of lung, trachea and endometrium of 22 of 32 rats that had exhibited clinical symptoms of mycoplasmal pneumonia prior to euthanasia, and whose organs had shown gross lesions suggesting mycoplasmal infection on necropsy, demonstrated the presence of Mycoplasma pulmonis organisms by the immunoperoxidase technique in one or more of these tissues.

In vivo and in vitro resistance to multiple anticholinesterases in Xenopus laevis tadpoles.

Natural and man-made anticholinesterases comprise a significant share of the Xenobiotic poisons to which many living organisms are exposed. To evaluate the potential correlation between the resistance of acetylcholinesterase (AChE) to such toxic agents and the systemic toxicity they confer, we characterized the sensitivity of AChE from Xenopus laevis tadpoles to inhibitors, examined the susceptibility of such tadpoles to poisoning by various anticholinesterases and tested the inhibitor sensitivities of recombinant human AChE produced in these amphibian embryos from microinjected DNA. Our findings reveal exceptionally high resistance of Xenopus AChE to carbamate, organophosphate and quaternary anticholinesterases. In spite of the effective in vivo penetrance to Xenopus tadpole tissues of paraoxon, the poisonous metabolite of the pro-insecticide parathion, the amphibian embryos displayed impressive resistance to this organophosphorous agent. The species specificity of this phenomenon was clearly displayed in Xenopus tadpoles expressing recombinant human AChE, which was far more sensitive than the frog enzyme to in vivo paraoxon inhibition. Our findings demonstrate a clear correlation between AChE susceptibility to enzymatic inhibition and the systemic toxicity of anticholinesterases and raise a serious concern regarding the use of Xenopus tadpoles for developmental toxicology tests of anticholinesterases.

Diagnosis and classification of amyloidosis by an immuno-histological method.

Following the recent classification of amyloidosis by the amyloid proteins involved, we decided to approach its tissue diagnosis and chemical classification by an immunohistological method. Post-mortem tissue specimens from various organs of patients with primary and secondary amyloidosis were examined by the immunoperoxidase technique with specific antibodies against proteins AL and AA which respectively characterize primary and secondary amyloidosis. The major advantage of this technique is that it can be applied to formalin fixed, paraffin embedded tissues. The immunoperoxidase technique proved to be extremely sensitive for the detection of amyloid deposits. Moreover, the deposited proteins could be clearly characterized as AL or AA by the specific anti-sera. We therefore believe that the immunooperoxidase technique is extremely useful for the early tissue diagnosis and chemical classification of amyloidosis.

Mucolipidosis IV: ultrastructural diagnosis of a recently defined genetic disorder.

In nine cases of a new genetic disorder mucolipidosis IV clinical findings included early or congenital corneal cloudiness and mild to severe psychomotor retardation in all patients. The diagnosis can be established by electron microscopy of the conjunctiva, which along with several other tissues shows typical ultrastructural changes. All of the known patients described were descendants of parents of Jewish-Ashkenazi origin, and it seems that mucolipidosis IV is another "Jewish" genetic disease. Prenatal diagnosis can be made by finding the typical ultrastructural changes in cells of the amniotic fluid. Biochemical abnormalities are inconspicuous, but there is evidence that the storage materials are gangliosides and hyaluronic acid.

Fine structure of Sertoli and Leydig cells in azoospermic human testis.

Testicular biopsy specimens taken from seven patients with azoospermia were studied by electron microscopy. We considered the possibility of a relationship between the annulate lamellae in the Sertoli cells and azoospermia. In the Leydig cells, we observed intranuclear and intracytoplasmic paracrystalline inclusions and considered their relationship with the Reinke crystalloid.

PIP: Special attention was given to the ultrastructure of 2 organelles: the annulate lamellae in the Sertoli cells and the crystalloid structure visible in the Leydig cells. Materials studied were biopsy specimens from 7 31-41 year old cases of male infertility with azoospermia or severe oligospermia. All biopsy specimens were bilateral. By light microscopy tubular atrophy with fibrosis and thickening of the boundary layer was observed. changes in the tubuli were characterized by an arrest of spermatogenesis at the stage of priamry spermatocyte production. Hyperplasia of Sertoli cells was noted with many fat vacuoles in the cytoplasm. Scattered large Sertoli cells contained Reinke crystalloids. By electron microscopy, large and irregular Sertoli cells contained abundant intracytoplasmic organelles and a prominent nucleus. The subcellular structures of the cytoplasm were all observed to be present. A specific organelle, the annulate lamellae or lamellar body, was seen in tight correlation with other structures near the nucleus. Other structural details observed are noted. Photographic reproductions illustrate electron microscopic findings. Although the presence of paracrystalline material is not specific to priamry testicular failure, the prominence of this structure in azoospermic human testes could be an expression of some modifications in the male hormone metabolism. It is suggested that a local estrogenic hyperaction could be the cause of depressed spermatogenesis and thus lead to azoospermia.

An immunoperoxidase study of cytomegalovirus mononucleosis.

Indirect immunoperoxidase method was used to evaluate retrospectively a case of cytomegalovirus (CMV) mononucleosis. Specific rabbit antiserum against CMV was used as primary reagent in routinely prepared paraffin sections of lymph node and in fixed slide preparations of the bone marrow aspirate. Staining specific for CMV antigen was observed in the lymphoid cells of the lymph node in the predominantly T-cell area and in the large lymphoid cells of the bone marrow. Bone marrow lymphoid cells with morphologic characteristics similar to those of the cells positive for CMV antigen showed positive immunoperoxidase reaction with specific rabbit antihuman T-cell serum. The data strongly indicate that CMV antigen was localized predominantly in the cells of lymphoid origin during the acute stage of the CMV mononucleosis; some data also suggest that infected lymphoid cells may be of T-cell origin. An immunoperoxidase technique is advantageous in both diagnosing and evaluating CMV mononucleosis.

Ultrastructural abnormalities of the liver in total lipodystrophy.

We present the results of light and electron microscopy studies of the liver in an 8-year-old girl with congenital total lipodystrophy. Liver histology revealed cirrhosis, and ultrastructural study showed mitochondrial abnormalities and an increase in the number of peroxisomes. A potential relationship between the high fatty acid concentration in the serum and the peroxisomal proliferation is considered.

Male pseudohermaphroditism due to 5 alpha-reductase deficiency. Ultrastructure of the gonads.

We report here the results of the endocrine studies and the morphological findings of the gonads in two male pseudohermaphrodite siblings with 5 alpha-reductase deficiency. Basal levels of luteinizing hormone, follicle-stimulating hormone, and prolactin were elevated and rose to very high levels in response to gonadotropin-releasing hormone and thyrotropin-releasing hormone,, when compared to those in normal males, while thyroid-stimulating hormone response was normal. Serum testosterone levels were within the normal range, and estradiol values were modestly elevated. Diagnosis was confirmed by lack of conversion of 3H-testoterone to 3H-dihydrotestosterone by fibroblasts taken from perineal skin. Pathologic examination of the testes in both patients disclosed tubular atrophy and complete arrest of spermatogenesis, with numberous hyperplastic Leydig cells containing large Reinke crystalloids.