Primary cutaneous B-cell lymphoma is associated with somatically hypermutated immunoglobulin variable genes and frequent use of VH1-69 and VH4-59 segments.

BACKGROUND: Accurate assessment of the somatic mutational status of clonal immunoglobulin variable region (IgV) genes is relevant in elucidating tumour cell origin in B-cell lymphoma; virgin B cells bear unmutated IgV genes, while germinal centre and postfollicular B cells carry mutated IgV genes. Furthermore, biases in the IgV repertoire and distribution pattern of somatic mutations indicate a possible antigen role in the pathogenesis of B-cell malignancies. OBJECTIVES: This work investigates the cellular origin and antigenic selection in primary cutaneous B-cell lymphoma (PCBCL). METHODS: We analysed the nucleotide sequence of clonal IgV heavy-chain gene (IgVH) rearrangements in 51 cases of PCBCL (25 follicle centre, 19 marginal zone and seven diffuse large B-cell lymphoma, leg-type) and compared IgVH sequences with their closest germline segment in the GenBank database. Molecular data were then correlated with histopathological features. RESULTS: We showed that all but one of the 51 IgVH sequences analysed exhibited extensive somatic hypermutations. The detected mutation rate ranged from 1.6% to 21%, with a median rate of 9.8% and was independent of PCBCL histotype. Calculation of antigen-selection pressure showed that 39% of the mutated IgVH genes displayed a number of replacement mutations and silent mutations in a pattern consistent with antigenic selection. Furthermore, two segments, VH1-69 (12%) and VH4-59 (14%), were preferentially used in our case series. CONCLUSIONS: Data indicate that neoplastic B cells of PBCBL have experienced germinal centre reaction and also suggest that the involvement of IgVH genes is not entirely random in PCBCL and that common antigen epitopes could be pathologically relevant in cutaneous lymphomagenesis.

Decreased c-Src expression enhances osteoblast differentiation and bone formation.

c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src(-/-) relative to Src(+/+) mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by approximately 60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src(-/-) osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice.

Association of primary varicose veins with dysregulated vein wall apoptosis.

BACKGROUND: Disordered programmed cell death may play a role in the development of superficial venous incompetence. We have determined the number of cells in apoptosis, and the mediators regulating the intrinsic and extrinsic pathways in specimens of varicose vein. METHODS: Venous segments were obtained from 46 patients undergoing surgical treatment for primary varicose veins. Controls samples were obtained from 20 patients undergoing distal arterial bypass grafting surgery. Segments of the distal and proximal saphenous trunk as well as tributaries were studied. Cell apoptoses and mediators of the mitochondrial and trans membrane pathway were evaluated with peroxidase in situ apoptosis detection, Bax and Fas detection, caspase-9 and 8 detection in the medial layer. RESULTS: Disorganised histological architecture was observed in varicose veins. Primary varicose veins also contained fewer peroxidase in situ-positive cells than control veins (2.6% S.D. 0.2% versus 12% S.D. 0.93%, P=.0001, Mann-Whitney u test), fewer Bax positive cells (2.1.% S.D. 0.3% versus 13% S.D. 0.9%, P=.0001) and fewer Caspase 9 positive cells (3.2% S.D. 1% versus 12% S.D. 1.3%, P=.0001). Similar findings were observed in saphenous trunk, main tributaries and accessory veins. In patients with recurrent varicose veins in whom the saphenous trunk had been preserved showed similar findings to primary varicose veins. Residual varicose veins contained fewer peroxidase in situ-positive cells than healthy veins (3.2% S.D. 0.6% versus 11% S.D. 2%, P=.0001), fewer Bax positive cells (2.2% S.D. 0.3% versus 12% S.D. 0.7%, P=.0001) and fewer Caspase 9 positive cells (2.6% S.D. 0.6% versus 12% S.D. 1%, P=.0001). Immunohistochemical detection for Fas and caspase 8 remained equal was the same in the varicose vein and control groups. CONCLUSION: Apoptosis is down regulated in the medial layer of varicose veins. This dysregulation is attributable to a disorder of the intrinsic pathway and involves the great saphenous vein trunk, major tributaries and accessory veins. This process may be among the causes of primary varicose veins.

[Glomerulosclerosis: pathogenetic mechanisms and possibility of regression]. / La sclerosi glomerulare meccanismi patogenetici e possibilita' di regressione.

Glomerular sclerosis means an increase in the extracellular matrix of the glomerulus. It is a complex, heterogeneous phenomenon with multiple cellular and biochemical mechanisms and different morphological patterns, depending on a variety of local and systemic factors. The term as such does not indicate the quantity nor the type of components of the deposited matrix. The amount of extracellular matrix increases in three types of events: - matrix deposition in areas that were destroyed by a necrotizing process (scars following glomerular necrosis); - matrix deposition in glomerular regions where matrix is normally found (mesangium, glomerular basement membranes) as seen in diabetic nephropathy; - matrix deposition inside or around collapsing capillaries involving the entire glomerulus or a segment of it (focal segmental glomerular sclerosis with nephrotic syndrome). Knowledge of the diverse morphological patterns producing glomerular sclerosis and the cellular and molecular mechanisms involved is essential for obvious reasons: they represent the rationale for any therapy aimed at preventing or reducing the progression of sclerosis and can provide a starting point to determine which forms are, at least potentially, reversible.

Incidence and nature of tumors induced in Sprague-Dawley rats by gamma-irradiation.

In our previous studies carried out on inbred rats of the Sprague-Dawley strain (L. Gross and Y. Dreyfuss, Proc. Natl. Acad. Sci. USA, 76: 5910-5913, 1979), the tumor incidence was increased following irradiation (150 rads, 5 times, at weekly intervals), from 22 to 93% in females and from 5 to 59% in males. Experiments here reported suggest that 2 consecutive total-body gamma-irradiations of 150 rads each are sufficient to induce in rats the development of tumors, some malignant; 18 of 19 females (94.7%) developed tumors at an average age of 11.4 mo, and seven of the 14 males in this group (50%) developed tumors at an average age of 10.4 mo. In the second group, which received 3 consecutive gamma-irradiations, 20 of 23 females (86.9%) and 5 of 13 males (38.4%) developed tumors at average ages of 9.1 and 7.5 mo, respectively. In the third group, among rats which received 4 consecutive gamma-irradiations, 17 of 19 females (89.4%) and 4 of 12 males (33.3%) developed tumors at average ages of 9.4 and 10.5 mo, respectively. The etiology of tumors either developing spontaneously or induced by irradiation in rats remains to be clarified. Our attempts to detect virus particles by electron microscopy in such tumors or lymphomas have not been successful. As a working hypothesis, we are tempted to theorize that tumors or lymphomas developing spontaneously or induced by gamma irradiation in rats are caused by latent viral agents which are integrated into the cell genome and are cell associated, i.e., not separable from the rat tumor cells by conventional methods thus far used.

Interobserver variability on the histopathologic diagnosis of cutaneous melanoma and other pigmented skin lesions.

PURPOSE: To assess the interobserver agreement on the diagnosis and classification of cutaneous melanoma. MATERIALS AND METHODS: A set of 140 slides of cutaneous melanoma, including a small subset of benign pigmented skin lesions, were circulated to four experienced histopathologists. The kappa statistic for multiple ratings per subject was calculated using the method described by Fleiss. RESULTS: The kappa value on the diagnosis of cutaneous melanoma versus benign lesions was 0.61. There was some discordance on the diagnosis in 37 of 140 cases (26%). For the histopathologic classification of cutaneous melanoma, the highest kappa values were attained for Breslow thickness (kappa = 0.76) and presence of ulceration (kappa = 0.87). The agreement was generally poor for other histologic features, such as level of dermal invasion (kappa = 0.38), presence of regression (kappa = 0.27), and lymphocytic infiltration (kappa = 0.27). CONCLUSION: Our study suggests considerable disagreement among pathologists on the diagnosis of melanoma versus other pigmented lesions. Tumor thickness and presence of ulceration are the most reproducible histologic features of cutaneous melanoma.

[Fabry disease]. / La malattia di Fabry.

Anderson--Fabry's disease is an hereditary disease with an X-linked genetic transmission, caused by the congenital deficiency of the lysosomial enzyme alpha-galactosidase A. It is a rare disease, but the estimated 1 patient every 117.000 male population is increasing and this can be demonstrated by simplifying the dosage techniques for the enzyme. The clinical picture comes from the accumulation of glycosphingolipids in many organs, mainly vessels, heart, nervous tissue, kidney and sight. The histological lesion appears as damage to the lysosomial membrane with subsequent migration of the lipid corpuscles into the cytoplasm and the breakdown of metabolic cellular activities. Prior to the advent of enzyme replacement therapy, which is based on the administration of the recombinant enzyme, the course of the disease was certainly fatal (early death by ictus or ischemic cardiopathy, terminal kidney failure). Despite the positive results achieved, information obtained from the present observation research should be help to verify the validity of the enzyme replacement therapy.

[The renal pathology in light chain deposition disease]. / Il coinvolgimento renale nella malattia da deposizione di catene leggere.

Light Chain Deposition Disease (LCDD) is a relatively frequent renal disease associated with dysproteinemia. Although the light chain deposits can be widespread, the kidney is the most frequently involved organ, and renal involvement can dominate the clinical condition. The morphological features of LCDD can be recognized by light microscopy; however, the diagnosis can be made certain only by immunofluorescence microscopy, using antisera to kappa and lambda chains, and by electron microscopy.

Colony stimulating factor-1-induced osteoclast spreading depends on substrate and requires the vitronectin receptor and the c-src proto-oncogene.

The colony stimulating factor 1 (CSF-1) regulates osteoclastogenesis and bone resorption. Mutations in the CSF-1 gene cause an osteopetrosis characterized by the absence of osteoclasts. Mature osteoclasts respond to CSF-1 with inhibition of bone resorption and an increment of cell spreading. Herein we demonstrate that CSF-1-induced osteoclast spreading depends on the substrate the osteoclast interacts with and requires integrity of the vitronectin receptor and of the c-src proto-oncogene. Rabbit osteoclasts were allowed to attach to glass, serum, osteopontin, and bone substrates, and were treated with 10 ng/ml human recombinant CSF-1 for 4 h. In osteoclasts plated on glass, the cytokine induced 70% inhibition of bone resorption and 1.8-fold stimulation of cell spreading, without changes in podosome expression and microfilament array. In contrast, CSF-1 induced a 2.5-fold increase of osteoclasts showing filopodia, and a 9.5-fold increase of osteoclasts presenting lamellipodia, indicating that membrane motility was required for cell spreading. Osteoclasts plated on serum substrates showed a 50% reduction of spontaneous spreading. However, in this circumstance, CSF-1 still stimulated an increase of osteoclast area. In osteoclasts cultured on osteopontin substrate or on bone slices, an inhibition of CSF-1-induced osteoclast spreading was observed. To establish involvement of the vitronectin receptor and c-src proto-oncogene, cells were treated with the alpha vbeta3 integrin neutralizing antibody, LM609, or c-src antisense oligonucleotides, which reduced CSF-1-induced osteoclast spreading by 57% and 60%, respectively. The results demonstrate that CSF-1-induced osteoclast spreading requires both the vitronectin receptor and the c-src proto-oncogene and that this action is modulated by the adhesion substrata.

Progressive increase in telomerase activity from benign melanocytic conditions to malignant melanoma.

The expression of telomerase activity and the in situ localization of the human telomerase RNA component (hTR) in melanocytic skin lesions was evaluated in specimens from sixty-three patients. Specimens of melanocytic nevi, primary melanomas and subcutaneous metastases of melanoma were obtained from fifty-eight patients, whereas metastasized lymph nodes were obtained from five patients. Telomerase activity was determined in these specimens by using a Polymerase Chain Reaction-based assay (TRAP). High relative mean telomerase activity levels were detected in metastatic melanoma (subcutaneous metastases = 54.5, lymph node metastases = 56.5). Much lower levels were detected in primary melanomas, which increased with advancing levels of tumor cell penetration (Clark II = 0.02, Clark III = 1.1, and Clark IV = 1.9). Twenty-six formalin-fixed, paraffin-embedded melanocytic lesions were sectioned and analyzed for telomerase RNA with a radioactive in situ hybridization assay. In situ hybridization studies with a probe to the template RNA component of telomerase confirmed that expression was almost exclusively confined to tumor cells and not infiltrating lymphocytes. These results indicate that levels of telomerase activity and telomerase RNA in melanocytic lesions correlate well with clinical stage and could potentially assist in the diagnosis of borderline lesions.

Melanoma cells stimulate osteoclastogenesis, c-Src expression and osteoblast cytokines.

Malignant melanomas metastasise to the bone and enhance osteoclast bone resorption. We demonstrated that a 48-h-B16 melanoma cell conditioned media (B16CM) induced osteoclastogenesis in mouse bone marrow cultures, without the requirement of B16 cell-bone marrow cell co-culture. B16 cells transcriptionally expressed detectable levels of TGFbeta1, IL-6, M-CSF, GM-CSF and TNFalpha mRNAs, albeit to a lower extent compared with levels in osteoblasts, and failed to express PTHrP, OPGL, OPG and IL-1beta. Interestingly, B16CM greatly upregulated IL-1beta, IL-6 and GM-CSF, and modestly enhanced TNFalpha and OPGL mRNA expression in osteoblasts, suggesting a potential indirect stimulation of osteoclastogenesis via the osteogenic lineage. B16CM barely upregulated c-Fos, but strongly and time-dependently enhanced c-Src expression in the total bone marrow cultures during osteoclast differentiation. Moreover, c-Src expression was enhanced in differentiated and purified osteoclast preparations to higher levels than in stromal cells. In conclusion, melanoma induces osteoclast generation with a paracrine mechanism independent of cell-cell contact, specifically upregulating c-Src in osteoclasts and cytokine expression in osteoblasts.

Melanocytic nevi of palms and soles: a histological study according to the plane of section.

Melanocytic nevi of palms and soles (MNPS) cause diagnostic problems to dermatopathologists because they share histologic features with malignant melanoma (MM). Early MNPS frequently display a striate appearance, suggesting that, in this subset of nevi, both melanocytes and melanin might have a particular distribution in relation to dermatoglyphics. To verify this hypothesis, we undertook a histological study on 78 junctional MNPS sampled along a plane either perpendicular or parallel to dermatoglyphics. Histologic examination revealed symmetry in 56% of the lesions, circumscription in 60%, intraepidermal scatter of melanocytes in 79%, and melanin columns in 61%. Interestingly, comparison between histologic features of nevi sampled perpendicularly and those of nevi cut parallely to dematoglyphics showed that features of benignity, namely symmetry, circumscription and melanin columns, were significantly more frequent in lesions dissected along a perpendicular plane. Moreover, in 70% of perpendicular samples, intraepidermal scatter of melanocytes and melanin columns were strictly concentrated in furrows. Therefore, to avoid diagnostic pitfalls in the differentiation between junctional MNPS and MM, we strongly suggest to dissect MNPS along a plane perpendicular to skin markings. We hypothesize that mechanical stress can be responsible for concentration of intraepidermal scatter of melanocytes and melanin columns in skin furrows.

Rotary shadowing of collagen monomers, oligomers, and fibrils during tendon fibrillogenesis.

Collagen monomers, oligomers, and fibrillar structures were isolated from chick tendons at various stages of development and studied by rotary shadowing. Monomers of Type I collagen, solubilized in 0.15 M NaCl solutions, were mostly present as collagen, pN-collagen, and pC-collagen with few procollagen molecules. They did not form polymers, nor were they associated with a carrier. Dimers of fibrillar collagen molecules were arranged in a 4-D stagger, suggesting that this was the preferred molecular interaction for the initiation of collagen fibrillogenesis. Type XII collagen molecules were mostly free, but some were attached by their central globular domain to one end of free fibrillar collagen molecules. Tenascin and Type VI collagen were also identified. The fibril populations consisted of collagen and beaded structures. These fibrils consisted of beads (globular domains) about 23 nm in diameter, separated by a period about 27 nm in length. Beads were linked by filamentous structures. These beaded fibrils probably represent the microfibrils of elastin.

Lectin-peroxidase conjugate reactivity in normal human kidney.

The carbohydrate histochemistry of normal human kidney has been investigated by the use of four peroxidase-labeled lectins at the light and electron microscopic level. The results show that the lectin of Lotus tetragonolobus, specific for l-fucose, binds exclusively to the proximal convoluted tubules of the nephron. While peanut and soybean lectins, specific for D-galactose and N-acetyl-D-galactosamine, respectively, are confirmed to the collecting ducts, wheat germ lectin, specific for sialic acid and N-acetyl-D-glucosamine, stains several parenchymal structures, including the glomerular capillary wall, particularly its podocyte cell coat. Sialidase digestion reveals strong binding sites for peanut and soybean lectin in the glomeruli. At the ultrastructural level most of the binding is shown to be on the podocyte surface and within the lamina rara externa of the basement membrane. The technique represents a potentially very useful tool for the study of various pathological states in the kidney.

Expression of sialic acid on the alveolar surface of adult and fetal human lungs.

Lung alveoli are coated by a thin layer of extracellular material rich in anionic charges. The nature of this acid layer and its relationship to the phospholipid surfactant are not known. We investigated the possible presence of sialic acid groups by light and electron microscopy in tissues from normal fetal and adult lungs, using neuraminidase treatment followed by staining with the galactose-binding lectin from peanut, labeled with peroxidase. Our results showed that adult lung does not bear peanut lectin-reactive sites but that a very thin and distinct reactive layer becomes evident after neuraminidase treatment, especially on type II pneumocytes. In fetal lung, the entire surface of the developing respiratory tree is outlined by a strongly peanut lectin-reactive layer even if neuraminidase digestion is not performed. We conclude that the acid coat of the alveolar lining is in part composed of sialic acid residues and that sialic acid is added to the fetal lung as the alveoli mature.

Immunohistochemical localization of renin in the human kidney.

By using an antiserum to purified human renal renin, renin was localized immunocytochemically in the human kidney under normal and various pathological conditions by the unlabeled antibody enzyme light microscope: (LM) and protein A-gold colloid electron microscope (EM) procedures. In the normal kidney, renin was confined to the epithelioid cells of the afferent arteriole of the juxtaglomerular apparatus (JGA). These cells were small and few, and always in the immediate neighborhood of the glomerulus. Fine structural analysis showed renin only in the secretion granules of the epitheloid cells. All granules within a given cell were stained with comparable intensity. In cases of renal artery stenosis (ischemic kidney) and of Bartter's syndrome, renin-positive epithelioid cells were larger, showed increased staining intensity, and were often found along the afferent arteriole at some distance from the glomerulus. Again, by electron microscopic observation, renin was seen only in secretion granules of epithelioid cells. In all of the above pathologic cases, plasma renin activity was very high. However, in the other nephropathies studied, renin staining in the kidney resembled that seen in normal kidneys, even when levels of plasma renin activity were quite high.

Spatial arrangement of subepithelial deposits in lupus and nonlupus membranous nephropathy.

Subepithelial deposits are a common feature of idiopathic membranous glomerulonephritis (MGN) and lupus membranous glomerulopathy (LMGN). We investigated the spatial arrangement of immunoglobulin G (IgG) and C3c fraction of complement (C3c) in the immune deposits of MGN and LMGN with confocal laser scanning microscopy to correlate specific patterns of IgG-C3 interactions with different diseases. Ten patients with MGN and 8 patients with LMGN (World Health Organization class VB) were selected. A determination of the spatial arrangement of the two fluorochromes and the glomerular area occupied by each fluorochrome was performed for each case. Our results showed MGN specimens have an orderly distribution of IgG and C3c, with each deposit showing an outer ring of sole IgG. IgG was always more abundant than C3c (1,619 +/- 271 v 790 +/- 105 micrometer(2), P = 0.002). In LMGN, IgG and C3c were haphazardly arranged, with deposits made of C3c only and an outer ring of IgG only rarely present. Also, the relative amounts of the two antigens were variable, and two groups could be identified (group 1: IgG, 5,515 +/- 1,179 micrometer(2) v C3c, 4,810 +/- 1,174 micrometer(2); P = 0.02; group 2: IgG, 3,358 +/- 658 micrometer(2) v C3c, 4,047 +/- 740 micrometer(2); P = 0.03). Our data show that diffuse IgG capping of the subepithelial immune deposits is diagnostic of MGN. The absence of an orderly three-dimensional arrangement in LMGN deposits (ie, outer ring of IgG) is likely to render active complement components more readily available to inflammatory activities.

Ultrastructural investigation of circulating villous lymphoid cells: a tool in the differential diagnosis of splenic lymphoma with villous lymphocytes.

Ultrastructural examination of circulating lymphoid cells was performed in three cases of splenic lymphoma with circulating lymphocytes (SLVL) in order to define morphological features helpful to distinguish this lymphoma from hairy cell leukemia (HCL). The samples for ultrastructural investigation were obtained by Ficoll sedimentation from peripheral blood and routinely processed for electron microscopy. The ultrastructural features examined were: morphology of villi, morphology of nuclei, presence of nucleoli, distribution of heterochromatin, type of cytoplasmic organelles, presence of specific intracytoplasmic structures such as the ribosome-lamella complex, lysosome-like bodies and perinuclear microfibrils. Our results and a careful review of the literature seemed to confirm that SLVL has electron microscopic features typical enough to be relevant in the differential diagnosis with HCL.

Light- and electron-microscopic study of clear cell chondrosarcoma.

Clear cell chondrosarcoma is a recently recognized tumor of the bone. It has a low-grade malignant potential and characteristic histologic appearance. A case studied by light and electron microscopy showed considerable resemblance to chondroblastoma, but in addition, there were clusters of cells with clear cytoplasm containing glycogen and representing areas of well-differentiated chondrosarcoma. Electron microscopy demonstrated chondroid cells in various stages of differentiation from immature chondroblasts to mature chondrocytes.

Diagnostic relevance of peripheral blood immunocytochemistry in hairy cell leukaemia.

AIMS--(1) To assess the diagnostic relevance of peripheral blood immunocytochemistry in hairy cell leukaemia (HCL); (2) to compare the immunostaining of bone marrow biopsy specimens with bone marrow and peripheral blood cytospins; (3) to evaluate the sensitivity of the different markers used; (4) to identify the ultrastructural localisation of DBA.44 in HCL variant. METHODS--Immunoenzymatic staining procedures, immunoperoxidase and immunoalkaline phosphatase, were used with a panel of monoclonal antibodies directed to HCL associated antigens. Ultrastructural immunostaining was performed using colloidal gold conjugated antibodies. RESULTS--HCL showed strong cytoplasmic reactivity for CD22, CD25, CD103, DBA.44, kappa, or lambda light chains. Peripheral blood diagnostic hairy cells were found in all the cases with absolute counts ranging from 0.11 x 10(9)/l up to 6.4 x 10(9)/l and values increasing with the size of the spleen. A median of 36.5% of leukaemic cells was found in bone marrow aspirates and 70% in bone marrow trephine specimens. The monoclonal antibodies CD22 and DBA.44 showed the highest and the lowest percentage of positive hairy cells, respectively; this difference was statistically significant (p = 0.0025). Ultrastructural immunolabelling with DBA.44 showed a cytoplasmic membrane localisation of the antigen in one case of HCL variant. CONCLUSIONS--(1) Immunocytochemistry is a useful technique which enhances the accuracy of diagnosis in HCL; (2) peripheral blood immunocytochemistry is recommended because it highlights hairy cells in all cases; (3) CD22 appears to be the most sensitive of the markers tested; (4) ultrastructural analysis is a useful tool in selected cases of HCL variant.