Hubert J. Wolfe

C-Cell Hyperplasia Preceding Medullary Thyroid Carcinoma

Abstract Two sisters at risk for hereditary medullary carcinoma and having small but progressive increases of serum calcitonin in response to calcium infusion underwent thyroidectomy. The thyroid g...

Production of cytokines around loosened cemented acetabular components. Analysis with immunohistochemical techniques and in situ hybridization.

The chronic inflammatory response to wear particles from orthopaedic joint implants is believed to cause osteolysis and to contribute to prosthetic loosening. Previous in vitro experiments have demonstrated that particulate debris from joint implants causes cells in culture to release products that have been implicated in this pathological bone resorption. The purpose of the current study was to investigate the in vivo features of this complex process in patients who had had a total hip replacement. Membraneous tissue was obtained from the cement-bone interface of ten polyethylene acetabular components that had been revised for aseptic loosening in ten patients. The immunoperoxidase technique, which involves the use of specific antibodies for each cell type, showed that macrophages were the predominant cellular constituents but also that fibroblasts, many of which were not identified on plain histological study, were present and were actively producing collagen. T lymphocytes were present variably, but they generally composed less than 10 percent of the cells. Particulate debris (polyethylene, methylmethacrylate, and metal) was present in all membrane specimens but was intracellular only in macrophages and multinucleated giant cells. 35S-labeled nucleic-acid probes, complementary to human interleukin-1-beta and to platelet-derived growth-factor-2 messenger RNA (mRNA), were hybridized with serial tissue sections. Hybridization demonstrated interleukin-1-beta mRNA predominantly in macrophages, and not in fibroblasts or in T lymphocytes to any major extent. In contrast, immunolocalization demonstrated interleukin-1-beta protein on both macrophages and fibroblasts, suggesting that macrophages release interleukin-1-beta, which then binds to both fibroblasts and macrophages. Platelet-derived growth-factor transcripts were found in both macrophages and fibroblasts.

A prospective trial of colchicine for primary biliary cirrhosis.

Abstract We entered 60 patients with primary biliary cirrhosis in a double-blind randomized controlled trial to determine whether colchicine is therapeutically effective. Thirty patients had early disease (Stages 1 and 2), and 30 had advanced disease (Stages 3 and 4). Fifteen patients with early disease and 15 with advanced disease received colchicine (0.6 mg twice daily), and the remainder received placebo. Patients were studied about every two months; those remaining in the blind phase at two years underwent repeat liver biopsy and were then placed on open-label colchicine (0.6 mg twice daily). With a few exceptions, the results in patients with early disease were similar to those in patients with advanced disease; hence, data on patients in all stages were combined in the main analysis. During the two-year study period the colchicine-treated patients, as compared with the placebo-treated patients, had improvement in levels of serum albumin, serum bilirubin, alkaline phosphatase, cholesterol, and aminot...

In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes

SummaryIn situ hybridization studies with [32P] and [3H] labelled antisense RNA probes were undertaken to determine optimal methods of tissue fixation, tissue sectioning, and conditions of hybridization, and to compare the relative merits of the two different radioactive labels. The distribution of somatostatin mRNA in neurons of rat brain using a labelled antisense somatostatin RNA probe was employed as a model for these studies. The highest degree of sensitivity forin situ hybridization was obtained using paraformaldehyde fixation and vibratome sectioning. Optimal autoradiographic localization of mRNA was obtained within 7 days using [32P] labelled probes. However, due to the high energy emittance of [32P], precise intracellular localization of hybridization sites was not possible. [3H] labelled RNA probes gave more precise cellular localization but required an average of 18–20 days autoradiographic exposure. The addition of the scintillator, PPO, decreased the exposure time for the localization of [3H] labelled probes to seven days. We also report a method for combinedin situ hybridization and immunocytochemistry for the simultaneous localization of somatostatin in mRNA and peptide in individual neurons.

Coexpression of platelet-derived growth factor (PDGF) and PDGF-receptor genes by primary human astrocytomas may contribute to their development and maintenance.

The present studies investigated the expression of the two PDGF genes (c-sis/PDGF-2 and PDGF-1) and the PDGF-receptor b gene (PDGF-R) in 34 primary human astrocytomas. Northern blot analysis demonstrated the coexpression of the c-sis/PDGF-2 protooncogene and the PDGF-R gene in all astrocytomas examined. The majority of the tumors also expressed the PDGF-1 gene. There was no correlation between the expression of the two PDGF genes. Nonmalignant human brain tissue expressed the PDGF-R and PDGF-1 genes but not the c-sis/PDGF-2 protooncogene. In situ hybridization of astrocytoma tissue localized the expression of the c-sis and PDGF-R mRNAs in tumor cells. Capillary endothelial cells also expressed c-sis mRNA. In contrast, nonmalignant human brain tissue expressed only PDGF-R mRNA but not c-sis/PDGF-2 mRNA. The coexpression of a potent mitogenic growth factor protooncogene (c-sis) and its receptor gene in astrocytoma tumor cells suggests the presence of an autocrine mechanism that may contribute to the development and maintenance of astrocytomas. The expression of c-sis mRNA in tumor cells but not in nonmalignant brain cells may serve as an additional diagnostic criterion for the detection of astrocytomas in small tissue specimen using in situ hybridization for the detection of c-sis mRNA and/or immunostaining for the recognition of its protein product.

Osteoporosis in Primary Biliary Cirrhosis: Effects of 25-Hydroxyvitamin D3 Treatment

Bone histology, bone mineral content, and calcium absorption were evaluated in 10 patients with primary biliary cirrhosis and osteopenia, before and after 1 yr of treatment with oral 25-hydroxycholecalciferol. Before treatment, quantitative histomorphometric analysis of full-thickness iliac crest bone biopsy specimens with double-tetracycline labeling demonstrated that 9 of 10 patients had osteoporosis. None had osteomalacia. Fasting intestinal calcium absorption correlated well with trabecular bone volume (r = 0.85). Bone mineral content measured by 125I-photon absorption was low in 6 of 10 patients, and it correlated poorly with iliac crest trabecular bone volume. After 1 yr of treatment with oral 25-hydroxyvitamin D3, bone mineral content fell in all 8 patients who were restudied. Iliac crest trabecular bone volume increased in 3 patients, 2 of whom had the greatest pretreatment impairment in calcium absorption, but fell in 5. Bone fractures continued to occur in 3 of 5 patients who were alive after 1 yr and developed for the first time in a sixth patient. We conclude that 25-hydroxyvitamin D3 is ineffective in reversing the bone thinning in the majority of primary biliary cirrhosis patients, but it may be helpful in a few selected patients.

Natural history of familial medullary thyroid carcinoma: effect of a program for early diagnosis.

To detect familial medullary thyroid carcinoma in a premetastatic stage, we administered tests provocative of calcitonin secretion (infusion of calcium or pentagastrin or both) each year for seven years to members of a pedigree now numbering 107. Since 1970, 21 patients converted from normal to abnormal secretory responses (two separate tests in which calcitonin levels exceeded 0.58 ng per milliliter). Twenty of 21 glands removed showed C-cell hyperplasia, and eight of the 20 also showed foci of carcinoma. As compared to the 12 patients with tumors detected during the first year of screening, all of whom had bilateral carcinoma (seven of 12 with local metastases), later carcinomas were smaller (mean diameter of 0.2 vs. 0.8 cm), were unilateral (in all but two cases) and occurred in younger patients (mean age of 14.9 vs. 36.4 years), and none had detectable metastases.

Distribution of Calcitonin-Containing Cells in the Normal Neonatal Human Thyroid Gland: A Correlation of Morphology with Peptide Content

C-cells have been mapped in the thyroid glands of 6 human neonates by means of immunoperoxidase localization of calcitonin and tissue calcitonin content as measured by radioimmunoassay. The C-cells were concentrated in a zone in the upper two-thirds of the lateral lobes bilaterally, where they were identified individually and in small groups in both an intrafollicular and parafollicular distribution. In contrast to findings in the adult, C-cells were predominantly intrafollicular in the neonate. The relative numbers of C-cells counted per unit area of thyroid tissue correlated strongly with the calcitonin content of immediately adjacent tissue sections. In areas rich in C-cells, as many as 75 immunoperoxidase-stained cells per low-power field were counted, and the concentration of calcitonin was as high as 540 to 2100 mU/g fresh weight, values that were as great as 10 times those observed in the normal adult thyroid gland. The prominence of the C-cell population and increased tissue calcitonin content in the human neonatal thyroid gland may reflect an as yet undefined physiologic role for calcitonin in the newborn.

Sustained Biochemical and Histologic Remission of Primary Biliary Cirrhosis in Response to Medical Treatment

Primary biliary cirrhosis is a chronic cholestatic liver disease that typically leads to liver transplantation or death [1]. The cause is unknown but is thought to be related to some inherited disorder of immunoregulation [2]. Although treatment with such drugs as ursodiol and colchicine may stabilize the disease or slow its rate of progression [3, 4], no reports on spontaneous or treatment-related remission of primary biliary cirrhosis have been published [5, 6]. Asymptomatic patients eventually develop symptoms and have shortened life expectancies; they are more appropriately called presymptomatic patients [7]. The introduction of methotrexate into the treatment of patients with primary biliary cirrhosis gives us cause to rethink this pessimistic out-look [8]. We describe five patients with well-documented primary biliary cirrhosis who have been treated with methotrexate for at least 6 years and whose disease has gone into remission while they received treatment. Methods Patients We focus on 5 of 19 patients with biopsy-proven precirrhotic primary biliary cirrhosis. Eighteen of the 19 patients were positive for antimitochondrial antibody, all had had primary biliary cirrhosis for at least 1 year, and all received low-dose methotrexate therapy. Nine of these patients have been described in an earlier report [8]. Three of the 5 patients we describe here had not responded to colchicine or had responded only partially. The 5 patients had received maintenance therapy with methotrexate for at least 6 years; the other 14 patients in the group had received methotrexate for 1 to 6 years. Intervention Methotrexate was given orally at a dosage of 15 mg/wk, divided into three 5-mg doses taken 12 hours apart over one 24-hour period. Histologic Studies Liver biopsy slides were coded and read independently by two senior pathologists who had no clinical information other than the fact that the patients had primary biliary cirrhosis. Biopsy specimens were paired by patient, but the pathologists were not aware of the timing of the biopsies (that is, initial compared with final). The length of the biopsy specimens and the number of portal triads on each slide were recorded. The specimens were graded according to histologic stage [9]; modified Knodell score, in which bile duct changes were graded and added to the total score [8]; and a score of global change (unchanged, 0; probably improved, +1; and unequivocally improved, +2) [4]. After each pathologist had read the slides independently, they met to resolve any differences and record the final histologic scores. Case Reports Patient 1 A 50-year-old woman was referred because of primary biliary cirrhosis that had developed 2 years earlier. The patient had been receiving cholestyramine, one packet three times per day, for 1 year. When primary biliary cirrhosis was first diagnosed, the alkaline phosphatase level was 750 IU (12.5 kat/L) and the patient was positive for antimitochondrial antibody (titer > 1:640). When the patient was seen at the New England Medical Center in Boston, results of physical examination were normal. At this point, the alkaline phosphatase level was 1245 IU (20.75 kat/L), the serum bilirubin level was 1.9 mg/dL (32.5 mol/L), and the alanine aminotransferase level was 235 U/L (3.9 kat/L). A liver biopsy specimen showed stage II to III primary biliary cirrhosis with many florid bile duct lesions (Figure 1, panel A). The patient was prescribed colchicine, 0.6 mg twice daily; cholestyramine therapy was continued. Figure 1. Liver biopsy specimens before and after methotrexate therapy for primary biliary cirrhosis. arrow During the next 5 years, the alkaline phosphatase level gradually decreased to 400 IU (6.67 kat/L), the serum bilirubin level decreased to 1 mg/dL (17.1 mol/L), and the alanine aminotransferase level decreased to 80 U/L (1.33 kat/L). The patient remained positive for antimitochondrial antibody. A repeated liver biopsy specimen showed decreased inflammation in portal triads but was still consistent with stage II disease. The patient began receiving oral methotrexate, 15 mg/wk. After the patient had received methotrexate for 1 year, cholestyramine therapy was discontinued; pruritus did not recur. After 1 year, during which the patient received the combination of methotrexate and colchicine, blood test results had improved: The alkaline phosphatase level was 200 IU (3.33 kat/L), the bilirubin level was 0.6 mg/dL (10.3 mol/L), and the alanine aminotransferase level was 50 U/L (0.83 kat/L). A repeated liver biopsy specimen showed only minimal portal fibrosis and inflammation. Because the alkaline phosphatase level was still elevated, the patient began receiving ursodiol, 300 mg twice daily. The alkaline phosphatase level decreased to normal during the next 12 months. After 3 years of treatment with all three medications, liver function test results were normal: The alkaline phosphatase level was 102 IU (1.7 kat/L), the bilirubin level was 0.5 mg/dL (8.5 kat/L), and the alanine aminotransferase level was 19 U/L (0.32 kat/L). A liver biopsy specimen obtained after 11 years of treatment (6 years of methotrexate therapy) was normal except for scattered foci of nonspecific inflammation in portal triads (Figure 1, panel B). The patient remains positive for antimitochondrial antibody after 14 years of treatment. Patient 2 A 34-year-old woman was referred because of a 1-year history of primary biliary cirrhosis. She reported pruritus and fatigue and was receiving hydroxyzine for pruritus. Results of physical examination showed striking bilateral xanthelasmas, diffuse excoriations, and an enlarged liver that was palpated 6 cm below the right costal margin. The serum alkaline phosphatase level was 962 IU (16 kat/L), the serum bilirubin level was 1.8 mg/dL (30.8 mol/L), and the alanine aminotransferase level was 251 U/L (4.2 kat/L). Results of tests for antimitochondrial antibody were positive (titer, 1:1024). A liver biopsy specimen showed stage III primary biliary cirrhosis with scattered florid bile duct lesions (Figure 1, panel C). The patient was prescribed oral methotrexate, 15 mg/wk. After 2 months of treatment, blood test results began to gradually but steadily improve. After 6 months of methotrexate therapy, the serum alkaline phosphatase level was 490 IU (8.2 kat/L), the serum bilirubin level was 0.7 mg/dL (12.0 mol/L), and the alanine aminotransferase level was 145 U/L (2.42 kat/L). After 8 months of therapy, pruritus and fatigue had disappeared and the patient could discontinue hydroxyzine therapy. Percutaneous liver biopsies done at years 1, 2, and 4 showed that inflammation and fibrosis had gradually diminished. After 4 years of methotrexate therapy, liver histologic findings were considerably improved. Scattered foci of inflammation were seen in portal triads, inflammation slightly extended into the periportal parenchyma, no portal-to-portal bridging inflammation or fibrosis was seen, and no florid bile duct lesions were present (Figure 1, panel D). The biopsy specimen was consistent with stage I primary biliary cirrhosis. Results of physical examination were normal. The xanthelasmas had disappeared, and the liver was no longer enlarged. After 6 years of methotrexate therapy, all liver function test results were normal: The serum alkaline phosphatase level was 94 IU (1.57 kat/L), the serum bilirubin level was 0.5 mg/dL (8.5 mol/L), and the alanine aminotransferase level was 17 U/L (0.28 kat/L). Results of tests for antimitochondrial antibody remained positive. The patient moved to another state and was lost to follow-up for 1.5 years. She discontinued methotrexate therapy and remained asymptomatic. After therapy was discontinued, however, the serum alkaline phosphatase level increased to 398 IU (6.63 kat/L) and the alanine aminotransferase level increased to 75 U/L (1.25 kat/L). The bilirubin level remained normal (0.3 mg/dL [5.1 mol/L]). The patient has resumed methotrexate therapy. Patient 3 A 42-year-old woman was referred because of primary biliary cirrhosis that had developed 4 years earlier. She had had the Raynaud phenomenon for 12 years. A maternal aunt had died of primary biliary cirrhosis. The patients serum alkaline phosphatase level was 263 IU (4.38 kat/L), the serum bilirubin level was 0.6 mg/dL (10.3 mol/L), and the alanine aminotransferase level was 57 U/L (0.95 kat/L). Testing by enzyme-linked immunosorbent assay showed strong positivity for antimitochondrial antibody: 0.35 OD (normal < 0.10 ODs) [10]. A percutaneous liver biopsy specimen revealed stage III primary biliary cirrhosis. The patient was prescribed colchicine, 0.6 mg twice daily. She remained asymptomatic, but blood test results did not improve. Repeated liver biopsy specimens obtained after 1 year showed worsening portal and periportal inflammation and the same degree of portal fibrosis (Figure 1, panel E). The patient began receiving oral methotrexate, 15 mg/wk, and remained asymptomatic. Two months after methotrexate therapy began, blood test results gradually improved. After 6 years of methotrexate and colchicine therapy, the serum alkaline phosphatase level had decreased to 96 IU (1.6 kat/L), the serum bilirubin level remained 0.6 mg/dL (10.3 mol/L), and the alanine aminotransferase level was 34 U/L (0.57 kat/L). Serial liver biopsy specimens, obtained at years 1, 2, 4, and 6 of methotrexate therapy, showed a steady decrease in inflammation and fibrosis. After 6 years of methotrexate and colchicine therapy, liver histologic findings are normal except for scattered foci of portal inflammation (Figure 1, panel F). Bile ducts are normal in appearance and number. The patient remains positive for antimitochondrial antibody. Results All five patients who responded biochemically and histologically had well-documented primary biliary cirrhosis that had been present for an average of 3.2 years (range, 1 to 7 years) before methotrexate therapy was started (Table 1). In e

In Situ Polymerase Chain Reaction Detection of Viral DNA, Single-Copy Genes, and Gene Rearrangements in Cell Suspensions and Cytospins

The study of low-copy viral or genomic DNA sequences by in situ hybridization (ISH) is often limited by sensitivity. Using the polymerase chain reaction (PCR) for the amplification of target DNA sequences in fixed cells [in situ PCR] (ISPCR) before ISH, we have been able to greatly improve the sensitivity of ISH. Viral DNA present in low copy number, single-copy genes, as well as immunoglobulin gene rearrangements (VH3 family genes), were successfully amplified in cells in suspension or on glass slides (cytospins). Single primer pairs were used in the in situ amplification step and 35S- or digoxigenin-11-dUTP-labeled region specific oligonucleotide probes were used for detection of amplificants by ISH. Artifacts, presumably resulting from leakage of in situ amplificants out of cells, may be a significant problem in selected instances. By optimal fixation and permeabilization of cells, limiting PCR cycle number, amplification of long DNA sequences, and/or incorporation of biotinylated dNTPs to produce bulkier amplificants together with washing of cells after ISPCR, diffusion artifacts were significantly reduced. Probe hybridization to single-stranded long PCR fragments or messenger RNA were excluded as a source for false-positive ISPCR results. The techniques reported dramatically increase the sensitivity of ISH in the detection of low-copy viral infection as well as in the study of gene rearrangements, and provide unique opportunities to study chromosomal translocations and point mutations at the cellular level.