Identification of titanium in human tissues: probable role in pathologic processes

Six cases of titanium dioxide exposure involving lung, skin, and synovium are described, with a review of the literature. The patients, four men and two women, were between the ages of 22 and 65 years. The pulmonary changes were characterized by fibrosis and numerous macrophages with abundant deposition of a black pigment. Adjacent areas of bronchopneumonia were also observed. In the skin a severe necrotizing lesion involving the subcutaneous tissue with extension to the muscle was observed in one case and a nonspecific inflammatory response was observed in another; both cases showed abundant black pigment deposition. Electron microscopy and energy dispersive x-ray analysis demonstrated the presence of large quantities of titanium in the pigment granules. There may be a combination of black pigment deposition and fibrosis, necrosis, or a xanthomatous or granulomatous reaction, that, together with negative results on special staining and culture studies for organisms, should raise the suspicion of titanium-associated injury and prompt the study of the affected tissues by x-ray analysis for positive identification.     

Identification of titanium pigment in drug addicts'' tissues

Autopsy on two drug addicts who had been injecting themselves intravenously with crushed Algafan tablets (active principle, propoxyphene hydrochloride) showed crystals consistent with talc in their lungs and other organs. A fine granular material that appeared black by transmitted light was also present. Electron microprobe analysis of an Algafan tablet showed an organic core and a coating that contained titanium, magnesium, silicon and calcium. Analysis of the crystals in the tissue sections showed magnesium and silicon in proportions compatible with talc, whilst fine granular material that probably represented the black pigment registered titanium. Titanium dioxide is a common whitening agent. It appears white by reflected light, black by transmitted light and pink by polarized light. This last feature should be helpful to histopathologists examining tissue sections containing titanium.     

Identification and significance of magnetite in human tissues

Magnetite or iron oxide has been identified in humans as well as certain animals and bacteria. With the current popularity of magnetic resonance imaging, the presence of these ferromagnetic particles in the tissues may impose biological significance. So far, identification of magnetite in tissue has been mainly based on magnetometry. Hence, a simple technique for direct identification of the magnetic particles in tissues is described. Lung tissues with abundant iron material and particles were digested in 1N sodium hydroxide solution. After rinsing, the sediments were suspended in 95% alcohol and placed on a glass slide located on a strong magnet. The iron-containing particles from the digestion procedure were aligned in a parallel manner along the north-south poles of the magnet and were confirmed to be magnetite by x-ray diffraction. No such effect was observed with hemosiderin-containing granules from the control liver tissues. The results of this experiment show that the "biological magnetite" is distinctly different from hemosiderin and has characteristic properties when subjected to a magnetic field.     

Identification and quantification of complement regulator CD46 on normal human tissues.

CD46 is a cell-surface regulatory molecule that prevents lysis of autologous human cells by activated complement. It has been well characterized on leucocytes, reproductive cells and various cultured cell lines and is considered to be ubiquitously expressed. We now extend these analyses and describe CD46 in a variety of different human tissues. Strong expression was observed by immunohistology on epithelial cells lining exocrine ducts and glands, such as salivary gland and pancreas and on kidney tubules and glomerular epithelium. Quantitative tissue expression was measured by radioimmunoassay and confirmed histological observations. Thus, CD46 is highly expressed on cells in contact with extracellular fluids thought not to contain large quantities of complement but which may still be subjected to complement attack thereby necessitating the presence of complement regulators to prevent non-specific destruction of cells.     

Distribution of interferon-gamma receptor in human tissues.

Interferon-gamma (IFN-gamma) is produced by activated T lymphocytes and plays a regulatory role in immune responses. The nature and location of cells that express the IFN-gamma receptor (R) and respond to this lymphokine are not well documented. The distribution of human IFN-gamma-R (HuIFN-gamma-R) was, therefore, investigated in situ by immunohistochemistry, using affinity-purified rabbit polyclonal antibodies directed against the extracellular domain of the receptor. In lymphoid organs, IFN-gamma-R expression is restricted to the B cell areas of lymph nodes, adult and fetal spleen, tonsils, appendix, and mucosa-associated lymphoid tissue of the small bowel. Macrophages and other reticular cells in lymphoid tissues and other organs are strongly positive for IFN-gamma-R, whereas its expression was consistently negative in the cortical and medullary thymocytes. Two-color flow cytofluorometric analysis of blood, lymph node, tonsil, spleen and thymus cells confirms that most B lymphocytes are IFN-gamma-R positive, whereas T lymphocytes are negative. However, after in vitro activation, peripheral blood T cells become IFN-gamma-R+. In non-lymphoid organs, IFN-gamma-R is expressed on endothelial cells of the medium- and small-size vessels. In epithelial tissues, high expression of IFN-gamma-R is detected on trophoblastic epithelium, glandular cells of stomach, ileum and colon, lung alveolar cells, salivary duct cells, renal tubular cells, and endometrial mucosa cells. Hepatocytes are weakly positive, while squamous epithelial cells are negative. The distribution of the HuIFN-gamma-R is discussed in view of the known functions of IFN-gamma.     

Identification of human papillomaviruses in paraffin embedded cervical pathological tissues from Indian women by polymerase chain reaction.

The polymerase chain reaction (PCR) technology was used to identify human papillomaviruses (HPV) in 52 paraffin embedded cervical tissues from Indian women with chronic cervicitis, different grades of cervical dysplasia and invasive cervical carcinoma. The tissues were screened for amplification of the cellular beta-globin gene as well as of HPVs. Sets of primers designed to amplify a portion of the E6 gene of HPV 6, 11, 16, 18, 31, 33 and 35 were employed. HPV 6, 16 and 31 were identified in 58% of 33 beta-globin positive tissues as compared to 16% of 19 beta-globin negative tissues. HPV 11, 18, 33 and 35 were not identified in any of the specimens. Double infection of HPV 16 and 31 was observed in one case of carcinoma in situ and one case of invasive carcinoma. HPV-16 was the predominant virus in HPV positive cases of higher grades of cervical dysplasia (severe dysplasia and carcinoma in situ) and cervical cancer.     

Dolichol and dolichyl phosphate in human tissues.

The content of dolichol and dolichyl phosphate in various human organs was analysed using autopsy samples. The reliability of these measurements was demonstrated by comparison with values for fresh biopsy material. Dolichol was present in all tissues investigated and the content was highest in the adrenal gland, pancreas, pituitary gland, testis and thyroid gland, ranging between 1.5 and 7.1 mg/g tissue. Dolichyl-P was detected in the various organs in highly variable amounts, ranging between 1 and 9% of the total dolichol content. While the main pattern of isoprene composition for dolichol and dolichyl-P was similar in individual organs, some variation was observed between tissues. Dolichol represents the largest lipid component in the pituitary gland, exceeding the total phospholipid content. The high concentrations of dolichol and dolichyl-P in human organs indicate that these lipids may play important roles in physiological and pathological cellular functions.     

Hamartin and tuberin expression in human tissues.

Tuberous sclerosis (TSC) is a bigenic autosomal dominant disease caused by mutations in one of two tumor-suppressor genes, TSC1 and TSC2, resulting in benign hamartomas and low grade neoplasms in multiple organs including brain, heart, kidney, and skin. We report the results of an immunohistochemical study of the expression of the TSC gene products, tuberin and hamartin, in multiple tissues obtained at autopsy from 12 non-TSC affected patients ranging in age from 20 weeks gestation to 8 years, and surgical specimens from some organs. Tuberin and hamartin are expressed and are colocalized in most tissues. Contrary to a previous report, immunostaining with our antisera detected hamartin in liver, small and large intestine, prostate, and testes. We did not detect significant developmental differences in tuberin or hamartin expression in comparable tissues from patients of different ages. Although tuberin and hamartin colocalize in most tissues and cell types, we provide data that hamartin is more abundantly expressed than tuberin in cells within some tissues including the distal nephron and a population of cells of the endocrine pancreas. These data support the hypothesis that hamartin and tuberin interact and may function together in many tissues where they are co-expressed, but also suggest that hamartin has a discrete and specialized function in certain cell types.     

Replication of influenza virus in organ cultures of human and simian urogenital tissues and human foetal tissues.

A survey of human adult tissues in organ cultures showed that influenza viruses (A/Moscow/1019/65 (h2n2) or a recombinant virus virulent for man (PR/8-A/England/939/69 Clone 7a(H3N2)) could infect uterus, bladder and conjunctiva but not oesophagus under the conditions employed; simian bladder and uterus were also susceptible. These results were similar to those already described for corresponding ferret tissues. Organ cultures of human foetal nasal mucosa, trachea, oesophagus, small and large intestine, and bladder consistently supported replication over 4 days or more with high virus yields. Lung, conjunctiva and umbilical cord were less consistently susceptible and gave lower yields. Placenta and kidney cultures allowed replication of virus in one of 8 and one of 4 experiments respectively, the yields being low and of short duration. Organ cultures of neural tissue (meninges and brain), lymphopoietic tissue (spleen, liver and thymus) and amnion did not support significant viral replication. The results are discussed in relation to possible infection of the foetus in utero with influenza virus.     

Immunohistochemical demonstration of leucocyte elastase in human tissues.

A polyclonal antiserum, produced in sheep and reactive against purified human leucocyte elastase, has been applied to paraffin sections from a range of human tissues by means of the indirect immunoperoxidase method. Striking and consistent results have been obtained. Normal or inflammatory granulocytes were intensely positive, the reaction being "blocked" by pure elastase. Activity was not seen in other sites, including histiocytic reticulum and lymphoid cells, although some weaker reaction was present in gastric and ileal lining epithelium. Strong reactivity was also seen in the cells of acute and chronic myeloid leukaemia and in extramedullary haemopoiesis. The advantages of this technique are compared with those for muramidase (lysozyme), alpha 1-antitrypsin, and naphthol-AS-D-chloroacetate esterase.     

Identification of acrylic cement particles in tissues

Investigation of the tissue response to acrylic cement particles is hampered by the fact that they are affected by tissue processing for transmission electron microscopy and for routine light microscopy. However, in the case of transmission electron microscopy, the present study shows that intracellular and extracellular cement sites can, in favourable circumstances, be identified by their heterogeneous appearance in the electron beam. This appearance results from the biphasic system which can form from the acrylic and the epoxy embedding medium during tissue processing. The validity of this means of identification has been confirmed (a) by observing the surface morphology of the particles preparedin vitro for the present study; (b) by use of barium sulphate as a marker in the cement; (c) by examination of control tissue samples. In the case of light microscopy, acrylic cement particles can be stained with Sudan dyes in frozen sections, as previously described by Willert and Semlitsch, or identified in paraffin sections by the presence of barium sulphate marker.     

Identification of Rickettsia rickettsii in formalin-fixed, paraffin-embedded tissues by immunofluorescence.

With slight modification of a trypsin digestion technique, Rickettsia rickettsii were demonstrated specifically by immunofluorescence staining in Formalin-fixed, paraffin-embedded tissue sections from a human, rhesus monkey, and guinea pig with Rocky Mountain spotted fever and in infected membranes from a chicken embryo. Tissues were cut at 4 micron and, using geltain as a tissue adhesive, were hydrated in a routine manner. Sections were then digested in refrigerated 0.1% trypsin for 16 h, washed, and stained specifically for R. rickettsii by direct or indirect immunofluorescence. Rickettsial organisms were localized in affected vessels of the mammalian species and within the yolk sac epithelium of the chicken embryo. Specificity was confirmed by adsorbing antibody conjugates with R. rickettsii organisms. Trypsin digestion probably decreased tissue proteins which interfered with immunochemical attachment of antibody to the rickettsiae. The technique is valuable in that a diagnosis of Rocky Mountain spotted fever can be confirmed from Formalin-fixed tissues processed in a routine manner.     

Beta2-microglobulin distribution in human normal tissues.

The distribution of beta2-microblobulin in human normal tissues was investigated by the indirect immunofluorescent antibody method. Lymphoid, macrophage and endothelial cells were consistently positive in every organ studied. In addition, only the stratum germinativum of the epidermis, some tracts of the columnar epithelium of the digestive system and some endometrial tubular glands showed a specific fluorescence.     

Identification in human lymphoid tissues of cells that produce group 1 or group 2 gamma-globulins

The cells that produce group 1 and group 2 γ-globulins have been localized in human lymphoid tissues. This has been done with the use of antisera specific for group 1 or group 2 γ-globulins prepared by immunizing rabbits with purified Bence-Jones proteins of the corresponding group and subsequently conjugated with different fluorochromes. The immunofluorescence observations have shown that in the red pulp of the spleen of adult humans two populations of plasma cells, present in approximately equal numbers, can be differentiated on the basis of the type of γ-globulin produced. The cells in the germinal centres of lymphoid follicles in the spleen and lymph nodes appear, instead, to contain both group 1 and group 2 γ-globulins.     

Biopterin derivatives in human body fluids and tissues.

Levels of biopterin derivatives in urine, serum, milk, cerebrospinal fluid, brain, and liver have been measured with the Crithidia fasciculata assay. Normal levels in serum and urine have been given and compared with those in a number of benign and malignant proliferative disorders, phenylketonuria, kidney disease, Parkinson's disease, schizophrenia, controlled epilepsy, rheumatoid arthritis, and pernicious anaemia. The active component of Crithidia factor in serum was 7,8-dihydrobiopterin. Tissue, urine, and some serum samples contained two active materials, the principal one being 7,8-dihydrobiopterin; a minor constituent was probably tetrahydrobiopterin. Serum biopterin levels following methotrexate administration were raised and subsequent administration of folic acid and 5-formyltetrahydrofolic acid further increased serum levels of biopterin derivatives; this was in contrast to the total absence of response to oral folates without prior methotrexate and to 5-methyltetrahydrofolic acid either with or without methotrexate being given.     

The distribution of immunoreactive interferon-alpha in normal human tissues.

The cellular distribution of immunoreactive interferon-alpha (IFN-alpha) was studied in formalin-fixed paraffin-embedded normal human tissues from 36 different organs. These samples were drawn from over 300 individuals none of whom had evidence of viral infection. Tissue histiocytes from all organs in the body, with the exception of brain and renal cortex and medulla, stained positively for IFN-alpha. Kupffer cells, pulmonary alveolar macrophages and lymph node macrophages were also positive. Parenchymal cells in some other organs also appeared to contain immunoreactive IFN-alpha. These included cuboidal lining cells of the choroid plexus in the brain, thyroid follicular cells, pituitary endocrine cells, adrenocortical cells and parathyroid principal and oxyphil cells. These findings are compatible with previous suggestions that IFN-alpha may be synthesized and released in the absence of viral infection and may thus have a role in normal physiology. The presence of IFN-alpha in most cells of the mononuclear phagocyte system suggests that these cells may play a major part in defence against viral infection.