Human Lyb-2 homolog CD72 is a marker for progenitor B-cell leukemias. S-HCL 2 is the prototype antibody of the recently defined CD72 cluster (human Lyb-2). Under nonreducing conditions, S-HCL 2 monoclonal antibody (mAb) precipitates a glycoprotein of 80-86 kDa. Under reducing conditions, a dimer of 43 and 39 kDa, with core proteins of 40 and 36 kDa, is precipitated. CD72 expression in normal and malignant tissues is different from expression of all other previously described human B-cell antigens. In peripheral blood and bone marrow, the antigen appears to be present on all B lymphocytes, with the exception of plasma cells. In tissue, immunohistochemical staining revealed positivity for all known B-cell compartments; however, pulpa macrophages of the spleen and von Kupffer cells exhibited distinct positivity for CD72 also. Among 83 malignant non-Hodgkin's lymphomas examined by immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase technique), all 54 B-cell lymphomas, including precursor B-cell lymphomas, Burkitt's lymphomas, germinal center lymphomas, chronic lymphocytic leukemias, and hairy cell leukemias, were CD72 positive, but no T-cell lymphomas were. Flow cytometry study of more than 80 mainly acute leukemias (52 B-cell leukemias) showed reactivity with S-HCL 2 mAb over the full range of B-cell differentiation. In particular, very early B cells in cytoplasmic Ig (cIg)-negative, CD19-positive pre-pre-B-cell leukemias and hybrid leukemias (mixed myeloid and B-cell type) were consistently positive for CD72 on the cell surface. Therefore, CD72 may become an important marker for progenitor B-cell leukemias.


Serum granulocyte colony-stimulating factor in patients with repeated infections. We have already reported significant elevation of serum granulocyte colony-stimulating factor (G-CSF) in the acute phase of infection. In this study, we compared the responses to infection between patients with frequently repeated infection (repeaters) and others (non-repeaters). We examined the clinical data and serum G-CSF levels in 48 patients with acute infections. Serum G-CSF levels were significantly lower in repeaters than in non-repeaters (197.7 +/- 370.0 vs. 1014.1 +/- 924.4 pg/ml. P less than 0.001). There were no significant differences in age, serum total protein, or cholinesterase between the groups, but serum albumin was significantly lower in repeaters than in non-repeaters (2.87 +/- 0.5 vs. 3.31 +/- 0.4 g/dl. P less than 0.005). It is suggested that administration of recombinant G-CSF may be useful for patients with repeated infections.


Sequential maturation stages of monoclonal B lineage cells from blood, spleen, lymph node, and bone marrow from a terminal myeloma patient. In order to fully understand the complexity of the monoclonal B lineage cells in multiple myeloma, it is necessary to evaluate the extent to which these cells are resident in solid lymphoid tissues and the phenotypic differences and similarities as compared to the circulating or bone marrow derived B lineage cells. Peripheral blood mononuclear cells from a patient with multiple myeloma were obtained 8 and 3 days prior to death, and mononuclear cells from lymph nodes, spleen, and bone marrow were obtained at autopsy. Rapid changes in the stage of differentiation of blood late-stage B lineage cells towards mature end-stage plasma cells were observed during the last week prior to death. Lymphoid cells within the blood comprised very few T cells, sub-normal numbers of monocytes, and 80% of B lineage cells which were at a late stage of differentiation. Shortly before death, plasma cells were found in the peripheral blood, indicating progression to plasma cell leukemia. At autopsy, the monoclonal B lineage cells in lymph node, spleen, and bone marrow represented different stages of terminal B cell differentiation. In each tissue, the B lineage cells were at an earlier differentiation stage, as defined phenotypically, than the circulating B lineage cells found in blood 3 days prior to death. Analysis of B cell markers and CD45 was used to define the differentiation stage of the relevant B cell populations, revealing a series of differentiation stages. The least mature B lineage cells (CD45hi) were found in lymph node. However, the CD45 isoform expressed was CD45R0, unlike most normal lymph node B cells. More differentiated B lineage cells (CD45med) were found in the bone marrow, and three sequential stages of pre-plasma cells were found in the spleen (CD45bright, CD45moderate, and CD45low-neg), all of which were CD45R0+. The B cells in normal spleen and bone marrow are CD45RA+. The presence of monoclonal B lineage cells in spleen was confirmed by Southern blotting. The B lineage cells from peripheral blood 3 days prior to death were approaching an end-stage plasma cell stage (CD45low/-). On B lineage cells from the various myeloma tissues, a concomitant loss of CD11b and increasing density of CD29 were observed as a function of progression to terminally differentiated stages.


Autoimmune neutropenia following peripheral blood stem cell transplantation. The differential diagnosis of unexpected neutropenia following bone marrow transplantation includes several potentially life-threatening complications including graft rejection, overwhelming infection, relapse of the underlying neoplasm, and intrinsic graft failure. However, a number of recent reports document that the differential diagnosis also includes autoimmune neutropenia, which, although potentially life-threatening, often responds well to corticosteroids or splenectomy. Autoimmune neutropenia has been reported following both autologous and allogeneic bone marrow transplantation. Herein we report a 31-year-old woman who developed a rapidly falling neutrophil count 11 days following peripheral blood stem cell transplantation for non-Hodgkin's lymphoma. A laboratory evaluation supported a diagnosis of autoimmune neutropenia, and the neutropenia resolved following treatment with steroids and granulocyte-colony stimulating factor.


Combined high-efficiency hemodialysis and charcoal hemoperfusion in severe N-acetylprocainamide intoxication. Several extracorporeal techniques have been used to remove N-acetylprocainamide (NAPA), the major metabolite of procainamide, in patients intoxicated with this substance. We report a patient with life-threatening NAPA intoxication who was rapidly and successfully treated with combined high-efficiency hemodialysis and charcoal hemoperfusion. The hemodialyzer and hemoperfusion cartridge were placed in series such that the patient's blood was dialyzed before reaching the cartridge. Overall clearance of NAPA was 153 mL/min, with clearance due to hemodialysis averaging 102 mL/min and that due to hemoperfusion averaging 88 mL/min. Thus, addition of the hemoperfusion cartridge into the extracorporeal circuit resulted in a 50% increase in clearance over that obtainable by high-efficiency hemodialysis alone. In comparison to other modalities, this technique is more effective than either hemodialysis or charcoal hemoperfusion alone and can achieve a more rapid reduction of serum NAPA levels than that observed with slow continuous hemofiltration or hemodiafiltration.


Mutation analysis of the cystic fibrosis transmembrane regulator gene in Native American populations of the southwest. We report DNA and clinical analyses of cystic fibrosis (CF) in two previously unstudied, genetically isolated populations: Pueblo and Navajo Native Americans. Direct mutation analysis of six mutations of the CFTR gene--namely, delta F508, G542X, G551D, R553X, N1303K, and W1282X--was performed on PCR-amplified genomic DNA extracted from blood samples. Haplotype analyses with marker/enzyme pairs XV2c/TaqI and KM19/PstI were performed as well. Of the 12 affected individuals studied, no delta F508 mutation was detected; only one G542X mutation was found. None of the other mutations was detected. All affected individuals have either an AA, AC, or CC haplotype, except for the one carrying the G542X mutation, who has the haplotype AB. Clinically, six of the affected individuals examined exhibit growth deficiency, and five (all from the Zuni Pueblo) have a severe CF phenotype. Four of the six Zunis with CF are also microcephalic, a finding not previously noted in CF patients. Our DNA data have serious implications for risk assessment of CF carrier status for these people.


Molecular analysis of mutations in a patient with purine nucleoside phosphorylase deficiency. Purine nucleoside phosphorylase (PNP) deficiency is an inherited autosomal recessive disorder resulting in severe combined immunodeficiency. The purpose of this study was to determine the molecular defects responsible for PNP deficiency in one such patient. The patient's PNP cDNA was amplified by PCR and sequenced. Point mutations leading to amino acid substitutions were found in both alleles. One point mutation led to a Ser-to-Gly substitution at amino acid 51 and was common to both alleles. In addition, an Asp-to-Gly substitution at amino acid 128 and an Arg-to-Pro substitution at amino acid 234 were found in the maternal and paternal alleles, respectively. In order to prove that these mutations were responsible for the disease state, each of the three mutations was constructed separately by site-directed mutagenesis of the normal PNP cDNA, and each was transiently expressed in COS cells. Lysates from cells transfected with the allele carrying the substitution at amino acid 51 retained both function and immunoreactivity. Lysates from cells transfected with PNP alleles carrying a substitution at either amino acid 128 or amino acid 234 contained immunoreactive material but had no detectable human PNP activity. In summary, molecular analysis of this patient identified point mutations within the PNP gene which are responsible for the enzyme deficiency.


A pseudodeficiency allele common in non-Jewish Tay-Sachs carriers: implications for carrier screening. Deficiency of beta-hexosaminidase A (Hex A) activity typically results in Tay-Sachs disease. However, healthy subjects found to be deficient in Hex A activity (i.e., pseudodeficient) by means of in vitro biochemical tests have been described. We analyzed the HEXA gene of one pseudodeficient subject and identified both a C739-to-T substitution that changes Arg247----Trp on one allele and a previously identified Tay-Sachs disease mutation on the second allele. Six additional pseudodeficient subjects were found to have the C739-to-T mutation. This allele accounted for 32% (20/62) of non-Jewish enzyme-defined Tay-Sachs disease carriers but for none of 36 Jewish enzyme-defined carriers who did not have one of three known mutations common to this group. The C739-to-T allele, together with a "true" Tay-Sachs disease allele, causes Hex A pseudodeficiency. Given both the large proportion of non-Jewish carriers with this allele and that standard biochemical screening cannot differentiate between heterozygotes for the C739-to-T mutations and Tay-Sachs disease carriers, DNA testing for this mutation in at-risk couples is essential. This could prevent unnecessary or incorrect prenatal diagnoses.


Pelizaeus-Merzbacher disease: detection of mutations Thr181----Pro and Leu223----Pro in the proteolipid protein gene, and prenatal diagnosis. A family with an apparent history of X-linked Pelizaeus-Merzbacher disease presented for genetic counseling, requesting carrier detection and prenatal diagnosis. RFLP analysis using the proteolipid protein (PLP) gene probe was uninformative in this family. A prenatal diagnosis on a chorionic villus sample (CVS) was carried out using single-strand conformation polymorphism (SSCP) analysis of a variant in exon 4 of the PLP gene. The fetus was predicted to be unaffected. Sequencing of the exon from the CVS, the predicted-carrier mother, and the obligate-carrier grandmother revealed an A-to-C change at nucleotide 541 in the two women but not in the fetus. As this change results in a Thr-to-Pro change at amino acid 181 in a region of the gene predicted to be part of a transmembrane segment, it was concluded that this was the mutation causing the disease in this family. In addition, in a second family, an exon 5 variant band pattern on SSCP analysis was shown by sequencing to be due to a T-to-C change at nucleotide 668. This results in a Leu-to-Pro change in a carrier mother and in her two affected sons. These results provide further examples of mutations in PLP that cause Pelizaeus-Merzbacher disease and illustrate the value of SSCP in genetic analysis.


Mutations of the KIT (mast/stem cell growth factor receptor) proto-oncogene account for a continuous range of phenotypes in human piebaldism. Piebaldism is a rare autosomal dominant disorder of pigmentation, characterized by congenital patches of white skin and hair from which melanocytes are absent. We have previously shown that piebaldism can result from missense and frameshift mutations of the KIT proto-oncogene, which encodes the cellular receptor tyrosine kinase for the mast/stem cell growth factor. Here, we report two novel KIT mutations associated with human piebaldism. A proximal frameshift is associated with a mild piebald phenotype, and a splice-junction mutation is associated with a highly variable piebald phenotype. We discuss the apparent relationship between the predicted impact of specific KIT mutations on total KIT-dependent signal transduction and the severity of the resultant piebald phenotypes.


Screening for five mutations detects 97% of cystic fibrosis (CF) chromosomes and predicts a carrier frequency of 1:29 in the Jewish Ashkenazi population. To determine the distribution and frequency of cystic fibrosis (CF) mutations in the Israeli population, we have screened 96 patients for 11 relatively common mutations. Five mutations--delta F508, G542X, W1282X, N1303K, and 3849 + 10kb C-->T--were found to account for 97% of the CF alleles in the Ashkenazi Jews. In contrast, of the 11 mutations tested, only delta F508 was detected in Jewish patients of Sephardic or Oriental origin, accounting for 43% of the CF alleles. Four mutations--delta F508, G542X, W1282X, and N1303K--accounted for 55% of the CF alleles in Arab patients. In a pilot screening study, a random sample of 424 Ashkenazi individuals was analyzed for three mutations--delta F508, W1282X, and G542X. Thirteen individuals were detected as heterozygotes (six for delta F508 and seven for W1282X), predicting a heterozygote frequency of 1:29. This is similar to the frequency of carriers in the Caucasian population of northern European ancestry. On the basis of these data, the Ashkenazi population is considered to be a candidate for CF heterozygote screening.


A molecular deletion of distal chromosome 4p in two families with a satellited chromosome 4 lacking the Wolf-Hirschhorn syndrome phenotype. We report two families with a satellited chromosome 4 short arm (4ps). Satellites and stalks normally occur on the short arms of acrocentric chromosomes; however, the literature cites several reports of satellited nonacrocentric chromosomes, which presumably result from a translocation with an acrocentric chromosome. This is the first report of 4ps chromosomes. Our families are remarkable in that both unaffected and affected individuals carry the 4ps chromosome. The phenotypes observed in affected individuals, although dissimilar, were sufficient to encourage a search for a deletion of chromosome 4p. By Southern blot analysis and fluorescence in situ hybridization, a deletion of material mapping approximately 150 kb from chromosome 4pter was discovered. This deletion is notable because it does not result in the Wolf-Hirschhorn syndrome and can result in an apparently normal phenotype. We speculate that homology between subterminal repeat sequences on 4p and sequences on the acrocentric short arms may explain the origin of the rearrangement and that position effect may play a role in the expression of the abnormal phenotype.


Case report: role of granulocyte colony stimulating factor in radiotherapy. Two patients who received granulocyte colony stimulating factor (G-CSF) before or during radiotherapy are reviewed. Both patients developed granulocytopenia after multi-agent chemotherapy. One patient had breast cancer with multiple distant metastasis and received palliative radiotherapy to the spine and hip joint. The other patient had locally advanced anal carcinoma and received concurrent 5-fluorouracil, mitomycin, and radiation therapy. Both patients completed the planned course of radiotherapy without interruption, after administration of G-CSF.


Unilateral radio-ulnar synostosis, generalized hypotonia, developmental retardation, and a characteristic facial appearance in sibs: a new syndrome. We report on 2 sibs with generalized hypotonia, developmental retardation, unilateral radio-ulnar synostosis, and a characteristic facial appearance. We propose that they have a new autosomal recessive syndrome.


Multiple-marker screening in pregnancies with hydropic and nonhydropic Turner syndrome. The combination of maternal serum alpha-fetoprotein, unconjugated estriol, and human chorionic gonadotropin levels and maternal age has been used to increase the sensitivity of screening for fetal Down syndrome and trisomy 18 in early-second-trimester pregnancies. We hypothesized that a unique pattern of these analytes also may be characteristic of fetal Turner syndrome, with or without hydrops. We studied preamniocentesis, second-trimester maternal serum specimens from seven hydropic and eight nonhydropic cases of fetal Turner syndrome. Clinical and pathologic records were reviewed. Statistical analysis of the data was performed by the rank sum test. In both hydropic and nonhydropic cases, alpha-fetoprotein levels were slightly reduced, and unconjugated estriol levels were markedly reduced. In hydropic pregnancies human chorionic gonadotropin levels were elevated, and nonhydropic pregnancies had low human chorionic gonadotropin levels (p = 0.001). The results suggest that the morphologic defect of hydrops, rather than the aneuploidy itself, is responsible for the elevation in human chorionic gonadotropin. In conjunction with the low unconjugated estriol levels, the elevation in human chorionic gonadotropin levels will result in the prenatal identification of hydropic fetal Turner syndrome pregnancies as being at increased risk for fetal Down syndrome.


Elevated maternal serum alpha-fetoprotein levels and midtrimester placental abnormalities in relation to subsequent adverse pregnancy outcomes. Unexplained elevations of maternal serum alpha-fetoprotein levels in the second trimester of pregnancy are associated with adverse pregnancy outcomes, including intrauterine growth retardation, preterm delivery, preeclampsia, and abruptio placentae. In addition, elevations of maternal serum alpha-fetoprotein have been associated with placental lesions detected during second-trimester ultrasonographic evaluations. We examined the relationship between adverse pregnancy outcomes and unexplained elevations of maternal serum alpha-fetoprotein and placental abnormalities in the second trimester of pregnancy. During the period from January 1989 to March 1991 we conducted a cohort study of 201 women with an elevated maternal serum alpha-fetoprotein (> or = 2.0 multiples of the median) and a second-trimester ultrasonographic evaluation at Swedish Hospital Medical Center and 211 women with normal maternal serum alpha-fetoprotein levels who had also undergone ultrasonographic evaluation at the same institution. All women in this investigation had singleton pregnancies without fetal anomalies. Elevated maternal serum alpha-fetoprotein was associated with the following adverse pregnancy outcomes: low birth weight (adjusted risk ratio 3.7), preterm delivery (adjusted risk ratio 3.6), intrauterine growth retardation (adjusted risk ratio 4.0), preeclampsia (adjusted risk ratio 3.8) and abruptio placentae (adjusted risk ratio 4.8). Placental abnormalities detected during second-trimester ultrasonographic evaluations were also associated with adverse pregnancy outcomes: low birth weight (adjusted risk ratio 2.0), preterm delivery (adjusted risk ratio 2.3), intrauterine growth retardation (adjusted risk ratio 1.4), and abruptio placentae (adjusted risk ratio 9.0). A joint positive history of second-trimester elevations of maternal serum alpha-fetoprotein and placental abnormalities was more strongly associated with the following adverse infant outcomes: low birth weight (adjusted risk ratio 6.9), preterm delivery (adjusted risk ratio 5.6), and intrauterine growth retardation (adjusted risk ratio 5.3). Unexplained elevated levels of maternal serum alpha-fetoprotein and placental abnormalities detected in the second trimester of pregnancy are associated with particularly poor pregnancy outcome. Careful examination for placental abnormalities should be part of the evaluation of elevated maternal serum alpha-fetoprotein.


Comparison of gram stain, leukocyte esterase activity, and amniotic fluid glucose concentration in predicting amniotic fluid culture results in preterm premature rupture of membranes. The purpose of this study was to prospectively compare three rapid and inexpensive tests that have been proposed as predictors of amniotic fluid culture results in preterm premature rupture of membranes. Amniocentesis was performed on 117 patients < or = 34 weeks' gestational age with premature rupture of membranes and no clinical evidence of infection. Amniotic fluid was sent for Gram stain and aerobic, anaerobic, and Mycoplasma cultures. Leukocyte esterase activity and glucose concentration were also determined on the amniotic fluid. Amniotic fluid cultures were positive in 56 patients (47.8%). Leukocyte esterase activity of 1+ or 2+ and an amniotic fluid glucose concentration < or = 16 mg/dl were significantly more sensitive (p < 0.005) than Gram stain in detecting positive amniotic fluid cultures (73%, 68%, and 41%, respectively). Although each of these rapid tests is useful in assessing for subclinical intraamniotic infection, none of them have sufficient accuracy to make clinical decisions solely on the basis of their results.


Midtrimester pregnancy termination: a randomized trial of prostaglandin E2 versus concentrated oxytocin. The purpose of this study was to determine whether a concentrated oxytocin infusion can reliably effect uterine evacuation in the midtrimester and whether such an infusion is associated with fewer side effects than prostaglandin E2 vaginal suppositories. Patients received either prostaglandin E2 (n = 42) or oxytocin (n = 45) for indicated midtrimester abortions in a prospective, randomized trial. Treatment consisted of either prostaglandin E2 vaginal suppositories (one every 4 hours) or infusions of an escalating concentration of oxytocin (one every 4 hours). Unless delivery had occurred or was imminent after 24 hours, the agent was considered to have failed, and patients were crossed to the alternative method. Delivery indications were similar between the two groups. There were 6 (14%) first-agent failures with prostaglandin E2 and 9 (20%) with oxytocin (p = 0.48). Considering the failures and subsequent crossovers, 103 patient trial regimens were completed. Fever, nausea, vomiting, and diarrhea were more frequent with prostaglandin E2 (p < 0.005). Concentrated oxytocin is a satisfactory alternative to prostaglandin E2 for midtrimester abortion.


Behr's syndrome and 3-methylglutaconic aciduria. We examined three patients from two families of Jewish-Iraqi origin who had progressive reduction of visual acuity and childhood onset of bilateral optic nerve atrophy without additional retinal abnormalities. They had neurologic symptoms compatible with Behr's syndrome. Neurologic signs included increased tendon reflexes, a positive Babinski sign, progressive spastic paraplegia, dysarthria, head nodding, and horizontal nystagmus. Neurologic involvement varied between affected siblings. The patients excreted excessive amounts of 3-methylglutaconic acid and 3-methylglutaric acid in their urine. We compared the characteristic ophthalmic features and the spectrum of neurologic signs encountered in this recently delineated autosomal recessive clinical entity with those of previously described entities associated with 3-methylglutaconic aciduria. Patients with early-onset optic atrophy should be examined for neurologic signs and screened for organic aciduria. A detailed ophthalmic examination is important in patients with neurologic abnormalities compatible with Behr's syndrome.


CD5 monoclonal antibodies react with human peripheral blood dendritic cells. CD5 monoclonal antibodies (MAbs) define a 67,000 kd monomeric glycoprotein expressed predominantly by thymocytes, mature T cells and a subpopulation of B cells. CD5 is believed to be an alternative signaling molecules capable of increasing the supply of second messengers and thereby altering the cellular response threshold to other activation stimuli. Human peripheral blood dendritic cells (PBDC) are a circulating component of the immune dendritic cell family, which also includes Langerhans' cells in epithelia and interdigitating cells in the T-cell domains of lymphoid tissues. PBDC comprise less than 1% of the peripheral blood mononuclear cell fraction. They are morphologically, immunophenotypically, and functionally distinct from monocytes. In this study, we report that at least a subpopulation of PBDC react with the anti-CD5 MAbs Leu-1 and UCHT2, which define the two major non-crossblocking CD5 epitopes. In contrast, Langerhans' cells, interdigitating cells, monocytes, and macrophages were uniformly CD5-. These findings suggest that PBDC can express the CD5 molecule. Furthermore, they define an additional feature of many enriched PBDC that distinguishes them from monocytes and certain other mononuclear leukocytes, and may provide insights into their activation pathways.


Markers for dysplasia of the upper aerodigestive tract. Suprabasal expression of PCNA, p53, and CK19 in alcohol-fixed, embedded tissue. Recognition of premalignant lesions in the oral epithelium has the potential to increase survival rates for squamous cell carcinoma of the oral cavity. It has previously been reported that cytokeratin 19 (CK19), a 40-kd epithelial cytoskeletal protein within the suprabasal squamous epithelium, is a specific marker of moderate-to-severe dysplasia and carcinoma in situ in oral cavity squamous epithelium. In contrast, normal epithelium and hyperplastic lesions reportedly express CK19 only in the basal layer if at all. The authors chose to test and extend this hypothesis by studying suprabasal CK19 expression and dysplasia of the oral cavity and upper aerodigestive tract in paraffin-embedded specimens that had been fixed in alcohol, a superior fixative for the preservation of cytokeratins. The authors examined 56 alcohol-fixed, paraffin-embedded specimens including 37 from the oral cavity, using two antibodies specific for CK19 (Ks19.1 and 4.62), an antibody to the nuclear proliferation marker, proliferating cell nuclear antigen (PCNA) (19A2), and an antibody to the putative tumor suppressor gene, p53 (pAb1801). The lesions were classified as normal, hyperplasia, mild dysplasia, moderate dysplasia, severe dysplasia/carcinoma in situ, or invasive squamous cell carcinoma, following standard histologic criteria. Immunocytochemically stained sections were scored for the presence or absence of suprabasal CK19, suprabasal PCNA, and p53 positivity, regardless of location. The immunostaining patterns of the two anti-CK19 antibodies were essentially equivalent. Except for one laryngeal specimen, normal epithelium, when positive, showed CK19 expression only in scattered cells throughout the basal layer. Proliferating cell nuclear antigen-positive nuclei were found exclusively in the basal layer. In areas of hyperplasia, CK19 immunostaining was absent or confined to the basal layer in 20 of 38 specimens and was expressed in suprabasal cells in 18 of 38 hyperplastic specimens. Proliferating cell nuclear antigen immunostaining in all cases of hyperplasia was limited to the basal layer. Severe dysplasia and carcinoma in situ showed suprabasal CK19 staining in six of nine specimens and no CK19 staining in three of nine specimens. In contrast, suprabasal PCNA immunostaining was found in all dysplasia and carcinoma in situ cases. p53 expression was detected in three of nine severe dysplasia/CIS specimens and was immunocytochemically undetectable in all normal, hyperplasia, and mild to moderate dysplasia specimens. The authors conclude that suprabasal CK19 expression is neither a sensitive nor a specific marker of premalignancy in oral epithelium and cannot be used to distinguish hyperplasia from dysplasia. In contrast, a strong correlation between suprabasal expression of PCNA, a marker for proliferating cells, and dysplasia/carcinoma in situ was evident.(ABSTRACT TRUNCATED AT 400 WORDS)


Role of beta 1-integrins in epidermotropism of malignant T cells. To better understand the molecular mechanisms of epidermotropism, we immunohistochemically analyzed the expression pattern of adhesion molecules belonging to the integrin and immunoglobulin superfamilies in cases of mycosis fungoides (MF) (n = 15), pleomorphic T cell lymphoma (n = 10), and high-grade T cell lymphoma (n = 7). The cutaneous T cell lymphomas (CTCLs) investigated were categorized into cases with or without epidermotropism. Focal neoexpression of ICAM-1 on keratinocytes was restricted to epidermotropic lymphomas. Both LFA-1 and LFA-3 were expressed on infiltrating cells in all cases investigated. In contrast, beta 1-integrins showed differential expression, most prominent in the case of VLA-1 and VLA-6: These molecules were present on infiltrating cells in most cases with epidermotropic MF and absent in most other CTCLs. We conclude that the phenomenon of epidermotropism might involve different sets of adhesion molecules in different entities of CTCL, with VLA-1 being the most influential beta 1-integrin in the case of MF.


Enhanced tenascin expression in cervical and vulvar koilocytotic lesions. Tenascin is an extracellular matrix glycoprotein that is widely expressed during embryogenesis. In adults, it is restricted to select sites, including certain epithelial-stromal interfaces, but is notably enhanced in active inflammatory-reactive processes and in the stroma of many neoplasms. The authors immunostained with the monoclonal antibody (MAb) 100EB2 cryosections of vulvar and cervical biopsies displaying convincing koilocytosis with variable degrees of hyperplasia-dysplasia; in situ carcinomas were included. The presence of human papillomaviruses (HPV) 6, 11, 16, 18, 31, or 33 was confirmed by in situ hybridization in a subset of cases. Findings were compared with normal controls. The study was extended with the MAb 143DB7 that reacted with tenascin in paraffin sections. In normal samples, tenascin immunoreaction appeared as a delicate, continuous rim, in the immediate vicinity of the laminin staining; in parakeratotic areas, the rim was thicker. In foci of hyperplastic-dysplastic epithelium with or without koilocytosis, a distinct increase in tenascin staining was noted; enhanced tenascin often paralleled increasing hyperplasia and dysplasia. In most cervical and vulvar carcinomas in situ, the reactions were intense and extended deeply and raggedly into the underlying stroma. Tenascin was selectively enhanced in the endocervical stroma around inflammed or metaplastic glands. The authors conclude that tenascin is increased in HPV infection associated with epithelial proliferation. Enhancement was most consistently strong and extensive in in situ carcinomas, suggesting a correlation with active phases of epithelial growth and stromal remodeling.


Nuclear translocation of monoclonal antibody directed against cell-surface carbohydrate Y determinant. Monoclonal antibody (MAb) Br 15-6A directed against the carbohydrate Y determinant expressed on tumor cells was found to be internalized and translocated to the nucleus of SK Br 5 breast carcinoma and SW 1116 and SW 707 colorectal carcinoma cells. Intracellular localization of MAb Br 15-6A was determined by cell fraction and by indirect immunofluorescence staining. Internalization of MAb Br 15-6A seems to be mediated by a specific cell surface protein of M(r) 108,000 in colorectal carcinoma cells and M(r) 92,000-96,000 in breast carcinoma cells. The MAb Br 15-6A precipitates an 88,000 M(r) chromatin protein and appears to be bound specifically to two EcoRI-digested and two HincII-chromatin fragments. Another MAb against the Y determinant (MAb Br 55.2) recognizes the same antigens as MAb Br 15-6A, but is not internalized.


Continuous secretion of monocyte chemotactic factors and fibroblast growth factors by alveolar macrophages following a single exposure to bleomycin in vitro. It has been shown in previous studies that alveolar macrophages incubated with bleomycin in vitro for 2 to 18 hours secrete monocyte chemotactic factors and fibroblast growth factors (MDGF). The purpose of the current experiments was to determine if alveolar macrophages similarly stimulated with bleomycin would continue to secrete these factors once the stimulus was removed. Alveolar macrophages from normal rats were exposed to bleomycin for 18 hours after which bleomycin was removed and macrophages maintained in culture for 35 days. Conditioned medium (CM) was collected and assayed at weekly intervals. In comparison with nonstimulated controls, bleomycin-stimulated macrophages secreted greater amounts of both monocyte chemotactic factors and MDGF for 35 days after exposure to bleomycin; with a significant difference noted between bleomycin and control macrophages for the first 21 days (P less than 0.02). In agreement with past work, the chemotactic activity in bleomycin-CM was due to fibronectin, as evidenced by the almost complete inhibition of activity by anti-fibronectin antibodies. The time course of secretion of chemotactic and growth factors after a single exposure to bleomycin in vitro was similar to that induced by in vivo exposure of macrophages to this drug. The data suggest that a similar direct activation of macrophages by bleomycin may promote the long-term production of these factors in vivo, resulting in continued monocyte recruitment and promotion of fibroblast proliferation in fibrotic lungs.


Possible role of interleukin 1 alpha and interleukin 1 beta in the pathogenesis of cholesteatoma of the middle ear. Cholesteatoma of the middle ear is characterized by the presence of hyperproliferative keratinizing squamous epithelium in the middle ear cavity and destruction of adjacent bone. Interleukin 1 (IL-1) is an autocrine growth factor for normal keratinocytes and is capable of inducing bone degradation. The distribution of two molecular species of IL-1, IL-1 alpha and IL-1 beta, was investigated immunohistochemically in the hyperproliferative epithelium of cholesteatoma, in normal epidermis of the auditory canal and of the retroauricular region, and in nonkeratinizing tonsillar epithelium. In all squamous epithelia examined, IL-1 alpha and IL-1 beta were present in comparable amounts. The IL-1 content of cholesteatoma epithelium was clearly increased in relation to normal skin keratinocytes. All cellular layers of cholesteatoma epithelium stained strongly and uniformly for Il-1 alpha and IL-1 beta, whereas the keratin layer was negative for IL-1. No particularly strong reaction with basal cells was detected. In the connective tissue under the squamous epithelium of cholesteatoma, intensely positive cells were scattered between negative stromal cells. Our results suggest that IL-1 could be liberated from disintegrating keratinocytes and cells of the monocyte-macrophage lineage, stimulate the proliferation of the cholesteatoma epithelium in an autocrine manner, and contribute to the enhancement of bone destruction in the presence of cholesteatoma.


Fetuin: its enigmatic property of growth promotion. A variety of cell types in culture respond to fetuin, a glycoprotein from fetal bovine serum, which is often an important supplement to many serum-free media. Bovine fetuin preparation has been shown to inhibit trypsin activity and promote cellular attachment, growth, and differentiation in many different culture systems. In addition, fetuin associates with various growth factors or growth-promoting substances. However, whether the growth-promoting activity of fetuin preparation is due to fetuin per se or to its minor contaminant(s) has been a long-standing puzzle. The present review surveys the literature concerning this enigmatic property of fetuin and summarizes three possibilities: 1) fetuin itself is active, although the majority of studies do not support this; 2) various contaminants of fetuin preparations, including potentially unidentified ones, are responsible for the activity, a possibility supported by numerous reports; and 3) one of the fetuin subspecies, one of its contaminants, or a combination of both of these is responsible for growth of a specific cell type. In addition, the basic physicochemical properties and other biological functions of fetuin have also been presented.


The iodide channel of the thyroid: a plasma membrane vesicle study. The uptake of radioactive iodide or chloride by plasma membrane vesicles of bovine thyroid was studied by a rapid filtration technique. A Na(+)-I- cotransport was demonstrated. When this Na(+)-I- cotransport is inactive (i.e., at 4 degrees C and in the absence of Na+), an uptake of iodide above chemical equilibrium could be induced, driven by the membrane potential. The latter was set up by allowing potassium to diffuse into the membrane vesicles in the presence of valinomycin and of an inward K+ gradient. This potential difference (positive inside) induced the uptake of iodide (or other anion present). The data support the existence of two anionic channels. The first one, observed at low near-physiological iodide concentration (micromolar range), which exhibits a high permeability and specificity for iodide (hence called the iodide channel), has a Km of 70 microM. The other one appears similar to the epithelial anion channel as described by Landry et al. (J. Gen. Physiol. 90: 779-798, 1987); it is still about fourfold more permeable to iodide than to chloride and presents a Km of 33 mM. Under physiological conditions the latter channel would mediate chloride transport, and the iodide channel, which is proposed to be restricted to the apical plasma membrane domain of the thyrocyte, transports iodide from the cytosol to the colloid space.


Evidence for an InsP3-gated channel protein in isolated rat olfactory cilia. Stimulation of rat olfactory cilia (ROC) with odorants leads to a transient elevation in the levels of either cAMP or inositol trisphosphate (InsP3). We have characterized the binding of [3H]InsP3 to isolated ROC. Unlabeled InsP3 displaced [3H]InsP3 binding in a dose-dependent manner (dissociation constant = 3.9 +/- 0.65 microM). Binding was stereospecific and dependent on the number of phosphates in the inositol ring. A ciliary protein of 120 kDa molecular mass was labeled specifically upon exposure of cilia membranes to ultraviolet light in the presence of the 125I-labeled InsP3 analogue 1-O-[N-(4-azidosaliciloxy)-3-aminopropyl-1-phospho]-myo-inositol 4,5-bisphosphate. Labeling of this protein displayed the same stereospecificity as binding of [3H]InsP3 to ROC. In addition, ROC membranes incorporated into a phospholipid bilayer at the tip of a patch pipette displayed an increase in conductance upon exposure to micromolar D-myoinositol 1,4,5-trisphosphate in 45% of the trials (n = 88). The InsP3-gated conductance is relatively nonspecific for cations and is distinct from the cAMP-gated conductance. The conductance displayed stereospecificity consistent with the InsP3 binding experiments. The results suggest that the site of action for odorant-stimulated elevations in InsP3 concentration in rat olfactory cilia is at a ciliary InsP3-gated channel.


Small linear chloride channels are endogenous to nonepithelial cells. We used both single-channel and whole cell patch-clamp techniques to characterize chloride channels and currents endogenous to Sf9 cells, 3T3 fibroblasts, and Chinese hamster ovary cells. In cell-attached patches from these cell types, anion channels were observed with low ohmic conductance (4-11 ps), linear current-voltage relationships, and little time- or voltage-dependent behavior. These channels are very similar to the Cl- channels reported to appear concomitant with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in these cell lines. The presence of such endogenous channels suggests either that low levels of CFTR are present in all of these cell lines prior to transfection or that an endogenous non-CFTR channel is present in these cell types. Our results suggest that at least some of the channel behaviors attributed to expressed, recombinant CFTR in previous studies may have been due to these endogenous Cl- channels.


Epitope mapping of monoclonal antibodies to bovine prolactin. The epitopes recognized by three monoclonal antibodies generated to sheep prolactin were determined by evaluating their cross-reactivities by immunodot analysis with 14 mutants of bovine prolactin, in which individual amino acids had been deleted or substituted. Mutations were made throughout the molecule and included disruption of the amino-terminal, carboxyl-terminal, and central disulfide loops. Lack of immunoreactivity was taken as an indication that the site of mutation was part of the epitope. Antibody 6F11 reacted with all bovine prolactin mutants tested, except those in which the carboxyl-terminal cysteine (position 199) was substituted by a serine. Antibodies 5G2 and 4C10 reacted with all of the bovine prolactin mutants, except those in which the amino-terminal cysteine (position 4) was substituted by a serine. Western blot analysis of sheep, squirrel monkey, and rat prolactins with the monoclonal antibodies revealed that 5G2 and 4C10 were specific for sheep prolactin, whereas antibody 6F11 cross-reacted with prolactins from all three species. The mitogenic activity of sheep or rat prolactin in the Nb2 bioassay was determined in the presence of the antibodies to determine whether the epitopes were part of the functional domains of these prolactins. The bioactivity of sheep prolactin (0.4 ng/ml) was unaffected by the monoclonal antibodies [0.01-1 microgram immunoglobulin G (IgG)/ml], whereas the bioactivity of rat prolactin (1.25 ng/ml) was inhibited by 6F11 with an apparent 50% inhibitory concentration of 0.25 microgram IgG/ml. These results indicate that monoclonal antibodies 5G2 and 4C10 cross-react with a species-specific region of the amino-terminal disulfide loop of bovine prolactin.(ABSTRACT TRUNCATED AT 250 WORDS)


Expression and localization of GLUT-5 in Caco-2 cells, human small intestine, and colon. The human colon carcinoma cell line Caco-2 was used as an enterocyte model to study the expression of the facilitative glucose transporters GLUT-1 and GLUT-2, and of the putative hexose transporter GLUT-5, which are expressed specifically in the gut. Northern blots indicate that Caco-2 cells express GLUT-1 and GLUT-5 mRNAs but not the mRNA coding for the basolateral glucose transporter GLUT-2. The level of GLUT-5 mRNA is growth dependent, being detectable only in postconfluent differentiated cells. In addition, the expression of GLUT-5 increases with the number of cell passages and is approximately 10 times higher in later passages (passage 184) than in early ones (passage 26). With the use of polyclonal antibodies directed against the COOH-terminus of GLUT-5, indirect immunofluorescence and Western blotting indicate that GLUT-5 is mainly localized to the brush border of Caco-2 cells. GLUT-5 is also found to be associated with the brush border of epithelial cells from fetal and normal adult human small intestine, but is absent from the colon.


Mechanism of action of cholecystokinin octapeptide on cat lower esophageal sphincter. In five cats we examined 1) the role of a cholinergic mechanism in cholecystokinin octapeptide (CCK-OP) excitation of the lower esophageal sphincter (LES) and 2) the interaction between CCK-OP-induced excitation and inhibition of the LES. Under ketamine anesthesia, LES pressure was monitored with a sleeve catheter. With CCK-OP, LES excitation was seen in four of five cats, and inhibition was seen in three of five cats. (4-Hydroxy-2-butynyl)trimethylammonium chloride (McNeil-A343) produced similar responses in the same cats but was less potent than CCK-OP. Atropine and/or hexamethonium reduced the response to CCK-OP. Pirenzepine had no effect on any CCK-OP response but reduced relaxation and enhanced excitation produced by McNeil-A343. Phentolamine increased CCK-OP-induced relaxation. In conclusion, CCK-OP can produce LES contraction through a preganglionic cholinergic mechanism involving a nicotinic synapse; however, induction of relaxation occurs predominantly at a postganglionic site involving adrenergic modulation. There is animal-to-animal variability in the balance of excitatory and inhibitory mechanisms to the LES, which determines the effect of a drug capable of activating both mechanisms.


Oxygen affects human endothelial cell proliferation by inactivation of fibroblast growth factors. The fibroblast growth factors (FGF), including endothelial cell growth factor (ECGF)/acidic FGF and basic FGF, are important modulators of endothelial cell replication in vitro and in vivo. Premature infants and adults with lung injuries are often treated with high levels of inspired O2, which can be necessary for survival but potentially injurious to developing lungs and in tissue repair following injury. Human umbilical artery and vein endothelial cells were grown in ECGF- or FGF-supplemented Medium 199 and exposed to ambient levels of O2 from 10 to 95%. Endothelial cell growth, measured by [3H]thymidine incorporation, was inhibited by increasing levels of O2 and ceased above 50% O2. Vein endothelial cells could recover from up to 24 h of hyperoxic exposure when given fresh medium, but not after 48 h. Artery-derived cells were more sensitive to O2 than were vein-derived cells. Complete medium without endothelial cells, preincubated 24 h in 95% O2, lost its ability to support cell growth under normoxic conditions. Exposing individual medium components to high O2 demonstrated that purified natural ECGF and recombinant acidic or basic FGF were all inactivated by O2. Human recombinant superoxide dismutase prevented FGF inactivation. O2 inactivation of essential growth factors could thus have major consequences for lung development or repair of injured capillaries in infants or adults inspiring high levels of O2.


The effect of dietary fat on diet selection may involve central serotonin. Rats consuming a diet of 34% tallow select more protein and less carbohydrate than rats fed either 5% corn oil or tallow or 34% corn oil (25). To examine potential mechanism(s) of this phenomenon, we fed rats diets containing either tallow or corn oil at levels of 5 or 34% for 2 days. Sera were analyzed, and rats fed 34% tallow had higher serum insulin compared with those fed 34% corn oil. In a second experiment, rats were fed either 34% corn oil or tallow for 2 days. Brain tissues were analyzed, and rats fed 34% tallow had elevated serotonin in the raphe area compared with those fed 34% corn oil. In a third experiment, rats were fed either 34% corn oil or tallow for 2 days and then given dl-fenfluramine before diet selection. Fenfluramine depressed food intake to a greater degree in rats fed 34% tallow compared with those fed corn oil. These findings suggest that the diet selection behavior observed in tallow-fed rats may be mediated by a central serotonin system.


Pathobiology of magnesium deficiency: a cytokine/neurogenic inflammation hypothesis. During the progression of Mg deficiency in a rodent model, we have observed dramatic increases in serum levels of inflammatory cytokines [interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha)] after 3 wk on a Mg-deficient diet. Sequential analyses of these cytokine changes in the serum of rats revealed an initial rise at day 12, followed by a major elevation in all three cytokine levels by day 21. Of greater interest was an early peak in the serum level of the neuropeptide substance P after only 5 days on the diet. This "neuronal" tachykinin is thought to be released from neural tissues, and it is known to stimulate production of certain cytokines, including IL-1, IL-6, and TNF-alpha. In addition, there was a concomitant increase in histamine levels, which may have resulted from stimulation and degranulation of mast cells by substance P. Thus we hypothesize that the release of substance P may be the earliest pathophysiological event leading to stimulation of the inflammatory cytokines, which may then stimulate the free radical mechanisms of injury previously confirmed by our work.


Substance P induces whole cell current transients in RBL-2H3 cells. To investigate the basis of interactions between nerves and mast cells, we tested the actions of the neuropeptide substance P (SP) on whole cell current characteristics of RBL-2H3 cells (homologous to mucosal mast cells). Control RBL cells showed a K(+)-dependent inwardly rectified current. SP (10(-6) M) caused transient, frequently repetitive increases in current amplitude, which at a membrane potential (Vm) of -80 mV rose by -1,020.0 +/- 223.4 pA after SP application compared with -6.8 +/- 1.7 pA for control. This response was characterized by a lag phase of 102 +/- 16 s. Seventeen percent of cells showed spontaneous transients in the current amplitude from the beginning of the recording. After SP administration, the amplitude of these transients increased by 6.3 +/- 2.0-fold. Responses to SP were mimicked by the application of ionomycin. For both SP and ionomycin, there was a dose dependency of the lag phase. Removal of extracellular calcium abolished the response for 10(-6) M SP but not for 6.6 x 10(-6) M ionomycin. During current transients, the whole cell current had both inward and outward rectified components with the zero current Vm shifted from -87.3 +/- 3.2 mV at control to -10.8 +/- 1.7 mV. We compare the SP-evoked current responses in mucosal-type mast cells with those described in connective tissue type.


Detection of angiotensin I and II in cultured rat cardiac myocytes and fibroblasts. Angiotensin II (ANG II) is a stimulus for positive chronotropic and inotropic effects, protein synthesis, and hypertrophic growth in cardiac tissue. These short- and long-term effects of ANG II are mediated through specific plasma membrane receptors. Indirect evidence suggests that ANG II synthesized in the myocardium may be important in regulating cardiac function. The cell types in the myocardium that produce components of the renin-angiotensin system have not been determined. In this study, we evaluated whether cultured cardiomyocytes and fibroblasts obtained from ventricles of neonatal rat hearts were capable of synthesizing ANG I and II. Both cardiomyocytes and fibroblasts were found to have immunofluorescent staining for ANG I, ANG II, and angiotensin-converting enzyme (ACE). The amounts of ANG I and II in cell extracts and conditioned media obtained from cardiomyocytes and fibroblasts were quantified by radioimmunoassay. The amounts of ANG I and II detected in cardiomyocyte cultures (1.48 x 10(6) cells/dish) were 32.2 +/- 16.2 (n = 4) and 6.2 +/- 2.9 (n = 4) ng/10(6) cells, respectively. The amounts of ANG I and II detected in the media conditioned by a 48-h exposure to cardiomyocytes were 5.2 +/- 1.2 (n = 3) and 2.1 +/- 1.2 (n = 3) ng/10(6) cells, respectively. The amounts of ANG I and II detected in fibroblast cultures (5.38 x 10(6) cells/dish) were 34.8 +/- 4.9 (n = 4) and 8.0 +/- 3.5 (n = 4) ng/10(6) cells, respectively. The amounts of ANG I and II obtained from media conditioned by a 48-h exposure to fibroblasts were 4.7 +/- 0.6 (n = 4) and 3.3 +/- 2.1 (n = 4) ng/10(6) cells, respectively. The identity of the radioimmunoassayable materials as ANG I and II peptides was confirmed in cardiomyocytes using an in vitro bioassay based on displacement of 125I-ANG II from receptor binding sites in cardiac membranes prepared from neonatal pig heart. Identification of ANG I and II and ACE in vitro in cultures of cardiac myocytes and fibroblasts supports the hypothesis that there is an intracardiac renin-angiotensin system that produces these peptides.


Glucocorticoid increases glucose cycling and inhibits insulin release in pancreatic islets of ob/ob mice. Normoglycemic ob/ob mice were treated for 24 or 48 h with either 25 micrograms/day of dexamethasone or saline. After an overnight fast, the animals were killed and pancreatic islets were incubated with 3H2O or [U-14C]glucose or [5-3H]glucose at 5.5 and 16.7 mM glucose. Incorporation of 3H from 3H2O into carbon 2 of medium glucose and the yield of 14CO2 from [U-14C]glucose and 3H2O from [5-3H]glucose were measured. Dexamethasone treatment for 48 h significantly increased the rate of dephosphorylation of glucose in islets both at 5.5 mM (24 vs. 16%) and 16.7 mM (56 vs. 36%) glucose, whereas glucose oxidation and utilization were unaffected. Dexamethasone treatment also inhibited insulin release by approximately 60% at 5.5 and 16.7 mM glucose, either in the presence or absence of 10 mM arginine, but had no effect when insulin release was stimulated by 1 mM 3-isobutyl-1-methylxanthine. Moreover, 24-h treatment with dexamethasone significantly increased glucose cycling at low and high glucose concentrations in the medium and inhibited insulin responsiveness to glucose and arginine. In conclusion, short-term dexamethasone treatment increases glucose flux through glucose-6-phosphatase in islets from ob/ob mice. This effect may contribute to the decreased insulin response to glucose and arginine found in animals treated with dexamethasone.


Gastrin induction of histamine release from primary cultures of canine oxyntic mucosal cells. Using enzyme-dispersed canine oxyntic mucosal cells, we studied regulation of histamine release from fractions in which mast cells were largely removed by density gradient. Histamine-like immunoreactivity was demonstrated using peroxidase-anti-peroxidase immunohistochemistry. Histamine-containing cells in the small cell elutriator fractions (SCEF) were further separated by albumin step density gradients. Approximately 2.5% of cells in the low density fraction (LDF) contained histamine-like immunoreactivity; this fraction was largely depleted of the more dense mast cells (0.5%). These two fractions were cultured for 48-64 h on a Matrigel substrate. The cell content of histamine and release into the medium were measured by radioenzymatic assay. Gastrin, carbachol, and forskolin increased histamine release from the LDF. The induction of histamine release by gastrin was evident within 5 min and was sustained for at least 60 min. The response to gastrin was dose dependent between concentrations of 10(-11) and 10(-8) M. In contrast, in the mast cell-enriched SCEF, basal release was higher and gastrin was without effect; however, concanavalin A stimulated and epinephrine inhibited histamine release indicating that histamine-release mechanisms were intact in this fraction. Our methods provide a preparation of low density oxyntic mucosal histamine cells that demonstrate gastrin-responsive histamine release; we speculate that enterochromaffin-like cells account for this gastrin response.


Galanin-induced alteration of electrolyte transport in the rat intestine. Effects of rat and porcine galanin on rat intestinal ion transport were examined in vitro. In the rat distal colon, a sustained increase in short-circuit current (Isc) was produced by the serosal addition of rat galanin at a concentration as low as 10(-9) M, and a maximal increment was observed at 10(-7) M. Porcine galanin was approximately 100 times less potent than rat galanin. In the rat jejunum, rat galanin produced only a slight and transient decrease in basal Isc. The response to rat galanin was not influenced by atropine, hexamethonium, or amiloride, but was virtually abolished by tetrodotoxin or furosemide. Rat galanin did not significantly influence the increase in Isc elicited by electrical field stimulation in the rat colon and jejunum. Transmural unidirectional 22Na and 36Cl fluxes in the rat colonic mucosa were measured under short-circuited conditions, and rat galanin significantly decreased net sodium and net chloride absorption. These findings suggest that galanin acts as a secretory modulator in the rat colon via noncholinergic neural transmission.


Toxicity of tubule fluid iron in the nephrotic syndrome. This study was carried out in rats with nephrotoxic serum nephritis after autologous phase proteinuria was well established to determine the effect of tubule fluid iron chelation on the course of this disease. Deferoxamine administration caused a reduction in urinary iron potentially capable of catalyzing hydroxyl radical (.OH) formation and kidney iron uptake (224 +/- 60 vs. 398 +/- 152 mg/kg). This was associated with a decrease rate of progression of renal failure over the 21-day study period (creatinine clearance -0. 199 +/- 0.152 vs. -0.509 +/- 0.336 ml/min, P < 0.05) and improved survival (8/8 vs. 4/8, P < 0.05). In addition deferoxamine caused a reduction in urinary transferrin excretion (32 +/- 15 vs. 74 +/- 16 mg/day) and fractional excretion of transferrin (2.01 +/- 1 vs. 5.9 +/- 3.7%) and an increase in serum transferrin levels (229 +/- 36 vs. 139 +/- 45 mg/dl, all P < 0.05). It is suggested that iron presented to the tubule fluid as a result of the glomerular leak for transferrin is dissociated from transferrin. In turn the iron is available in a form capable of catalyzing .OH formation, resulting in lipid peroxidation of tubule cell membranes. Deferoxamine chelation of tubule fluid iron retards the development of both tubulointerstitial injury and superimposed glomerular sclerosis in this model of membranous nephropathy.


L-selectin function is required for beta 2-integrin-mediated neutrophil adhesion at physiological shear rates in vivo. In vivo interactions between neutrophils and endothelial cells (EC) follow a multistep process involving two distinct neutrophil adhesion receptors. L-selectin, constitutively functional on resting neutrophils, mediates an activation-independent primary interaction resulting in rolling along the venular wall. Subsequent activation of rolling neutrophils induces upregulation and functional activation of beta 2-integrins (CD11/CD18) leading to firm attachment. Based on previous findings we hypothesized that, under shear force, rolling may be essential for successful neutrophil-EC recognition. Here we report results of our studies of human neutrophil behavior in interleukin (IL)-1-activated rabbit mesentery venules, an interaction that requires both L-selectin and beta 2-integrins. Rolling of human neutrophils is L-selection mediated; it was strongly reduced by monoclonal antibody inhibition or enzymatic removal of L-selectin. Furthermore, activation induced L-selectin shedding and, in a dose- and time-dependent fashion, rendered neutrophils unable to recognize inflamed EC despite expression of active beta 2-integrins, which promoted adhesion in vitro. Neutrophils activated for 5 min or longer lost most of their ability to roll. However, 1-3 min after activation, rolling was reduced (not abolished), and cells that were still able to roll displayed a significant tendency for a CD18-dependent transition from rolling to sticking. The whole sequence of events, rolling, sticking, and transendothelial migration, could be observed if an extravascular chemotactic stimulus was applied by superfusing mesenteries with leukotriene B4. Under such conditions, sticking and emigration was blocked when rolling was inhibited by enzymatic removal of L-selectin. Our results indicate that primary neutrophil interaction with inflamed EC through the L-selectin is a prerequisite for neutrophil function at physiological shear rates in vivo.


Mechanism of enhanced myogenic response in arterioles during sympathetic nerve stimulation. In a previous study we found that the arteriolar myogenic response was enhanced during sympathetic nerve stimulation in the cat sartorius muscle. In this study we determined whether the enhancement was unique to sympathetic nerve stimulation. Changes of arteriolar diameter and red cell velocity during femoral arterial pressure reduction from 110 to 60 mmHg were examined. Arterioles of 40 microns diameter were constricted by norepinephrine infusion to a similar degree as sympathetic nerve stimulation. Arteriolar dilation to pressure reduction was significantly enhanced during norepinephrine infusion and was not significantly different from that during sympathetic nerve stimulation. This indicates that junctional release of transmitters is not essential and rules out prejunctional inhibition of neurotransmitter release during pressure reduction as a significant mechanism in the enhanced dilation. Arteriolar dilation to pressure reduction was also enhanced during vasopressin or BAY K 8644 (a calcium channel agonist) infusion. In all instances, autoregulation of flow was significantly enhanced. These results demonstrate that modulation of the myogenic response occurs at postreceptor sites in the smooth muscle cell.


Recovery of anoxic-reoxygenated cardiomyocytes from severe Ca2+ overload. The ability of hypoxic-reoxygenated cardiomyocytes to recover from severe cytosolic Ca2+ overload was investigated using the fluorescent Ca2+ indicator fura-2 in ventricular cardiomyocytes from adult rats. When the fura-2 ratio (340/380 nm) reached saturation in hypoxic cardiomyocytes, indicating severe Ca2+ overload, they were reoxygenated. The cell then suddenly hypercontracted but reestablished, after a phase of Ca2+ oscillations, a normal Ca2+ control. Because these oscillations could be abolished by ryanodine (50 nM), they seem to depend on the function of the sarcoplasmic reticulum (SR). In the presence of caffeine (5 mM) and thapsigargin (100 nM), i.e., agents impairing Ca2+ sequestration in the SR, reoxygenation did not lead to Ca2+ oscillations or to a stable recovery of cytosolic Ca2+ control. The additional presence of ruthenium red (5 microM), an inhibitor of mitochondrial Ca2+ uptake, restored the ability of cells treated with caffeine or thapsigargin to reestablish a normal cytosolic Ca2+ control. The results show that cardiomyocytes are able to recover from severe hypoxic Ca2+ overload if, first, a closed sarcolemma is retained (as in isolated cardiomyocytes) and, second, the SR is available for rapid Ca2+ storage (impaired by caffeine and thapsigargin). The results also suggest that, in the case of an impairment of SR function, the inhibition of mitochondrial Ca2+ uptake (as by ruthenium red) has a protective effect.


Presence of C-type natriuretic peptide in cultured human endothelial cells and plasma. The present study was undertaken to investigate the presence of C-type natriuretic peptide (CNP) immunoreactivity in cultured human vascular endothelial cells and in human plasma. CNP immunoreactivity was present in cultured human aortic endothelial cells by both immunohistochemical staining and by radioimmunoassay. With the utilization of gel permeation chromatography, this immunoreactivity proved to be consistent with the higher molecular weight CNP-53. CNP immunoreactivity was also present in human plasma (n = 22) at low picogram concentrations (6.5 +/- 0.2 pg/ml) by specific radioimmunoassay. This immunoreactivity was consistent with the lower molecular weight CNP-22 by gel permeation chromatography. These findings suggest that the vascular endothelium may be the site of CNP production. The isolation of different molecular forms of CNP in tissue and plasma may be consistent with a storage form of the peptide in endothelial cells CNP-53, while CNP-22 circulates in plasma. In summary, the present study is consistent with CNP being a peptide of endothelial cell origin.


Technique for training schizophrenic patients in illness self-management: a controlled trial. To determine whether schizophrenic outpatients receiving low-dose neuroleptic therapy could learn and retain complex information and skills related to self-management of their illness, a novel technique of teaching, using cognitive and behavioral methods, was designed to compensate for the patients' learning disabilities. The subjects were 41 patients with DSM-III-R schizophrenia who were receiving constant maintenance neuroleptic drug therapy. They were randomly assigned to structured, modularized skills training or to supportive group psychotherapy. The patients who received skills training made significant gains in each of the areas taught, while those participating in group therapy did not. The skills learned during training were retained without significant erosion over a 1-year follow-up period. The effectiveness of modularized teaching of illness self-management skills to schizophrenic patients appears to be largely independent of baseline psychology and symptom improvement. Such an approach is useful for overcoming or compensating for the enduring cognitive and information processing deficits commonly found in schizophrenia.


The interchange of disease and health between the Old and New Worlds. A review of the five centuries since Columbus discovered America helps us understand the mutual contributions of the Old and the New Worlds to the history of diseases and their treatment. It also shows the consequences of this "mutual discovery" as they are currently emerging in the fields of health, culture, and the environment. To evaluate the multiple aspects of the interchange between the Old and New Worlds, this paper discusses the following: the causes of the rapid decline of the original American populations; the diffusion of communicable diseases between the two civilizations; the health consequences of nutritional changes on both sides of the Atlantic; drug addictions, as they developed through the centuries and as they exist today; the ways diseases were and are evaluated, prevented, diagnosed, and treated; and the mutual impact of different models of health services. Arguing that a major global change following the discovery of America was the transition from isolation of the two worlds to communication, and, more recently, to global interdependence, the paper also discusses some problems of bioethical relevance and the possible impact of new epidemics. Finally, it suggests that a critical analysis of the past may help stimulate future cooperation and solidarity.


Metastatic prostatic carcinoma presenting as an oncocytic tumor. We discuss a 63-year-old man who presented with a metastatic tumor in an inguinal lymph node. By light microscopy, the tumor cells were characterized by a finely granular eosinophilic cytoplasm. A diagnosis of metastatic oncocytic carcinoma was made based on the results of an ultrastructural examination, which showed the cytoplasm of the tumor cells to be filled with mitochondria. Results of immunocytochemical studies showed positive reactivity for prostatic acid phosphatase and prostate-specific antigen. A transurethral resection of the prostate showed an oncocytic adenocarcinoma of the prostate, apparently the first of its kind, which was demonstrated to be the site of origin of the inguinal lymph node metastasis.


Intraarticular synovial sarcoma.

Intraarticular synovial sarcoma. A 43-year-old man presented with decreased range of motion in his left knee and a painful medial joint mass that was grossly visible. Arthroscopy demonstrated a mobile, flat mass 3 cm in diameter in the knee joint that seemed to be loosely tethered to the synovium. The mass was excised, and light microscopic examination demonstrated a biphasic synovial sarcoma. There was no transition with the attached normal synovium. Immunohistochemically, the epithelial component was intensely positive for epithelial membrane antigen and cytokeratins (CAM 5.2 and AE 1/AE 3), and the spindle cell component was focally positive for these markers. The patient has no evidence of disease 9 years after only local excision. Although the term synovial sarcoma suggests a relationship to normal synovium, only rarely has truly intraarticular disease been reported.


Primary ovarian small cell carcinoma of pulmonary type. A clinicopathologic, immunohistologic, and flow cytometric analysis of 11 cases. Eleven primary ovarian tumors that resembled small cell carcinoma of the lung are reported. They occurred in women 28-85 (mean 59) years of age, most of whom presented with abdominal swelling. Six of the tumors were unilateral and five bilateral; seven had spread beyond the ovary. The tumors ranged from 4.5 to 26 (mean 13.5) cm in greatest dimension and were mostly solid, with a variable minor cystic component. Microscopic examination showed small to medium-sized round to spindle-shaped cells with scanty cytoplasm, hyperchromatic nuclei, and inconspicuous nucleoli growing in sheets, closely packed nests, and occasionally islands and trabeculae. A component of endometrioid carcinoma was present in four tumors, another tumor showed squamous differentiation, another contained a cyst lined by atypical mucinous cells, and two others were associated with a Brenner tumor. Argyrophil granules were present focally in two of six tumors appropriately stained. Immunohistochemical staining was performed in nine cases: in six there was staining of the small cell component for keratin, in five for epithelial membrane antigen, in seven for neuron-specific enolase, in two for chromogranin, and in one for Leu-7. Vimentin staining was not observed. Flow cytometry was performed on eight tumors: five were aneuploid and three diploid. Five of seven patients with long-term follow-up died of, or with, disease at 1-13 (mean 8) months, one died after an unknown interval, and one was alive at 7.5 years. Two other patients had recurrent or residual disease at 6 and 8 months.


Basaloid squamous cell carcinoma of the head and neck. A clinicopathologic and immunohistochemical study of 40 cases. In this study of 40 cases of basaloid squamous cell carcinoma, 83% arose in the pyriform sinus, base of tongue, tonsil, and larynx. The 35 men and five women ranged in age from 27 to 88 years (median 62). In patients for whom social habits were recorded, 24 of 26 patients were smokers and 22 of 25 drank ethanol. Most presented with stage III or IV disease. Twenty-seven patients had regional metastases at the time of presentation and 15 developed distant metastases. Seventeen patients died with disease (median survival 18 months). The tumors were composed of moderately pleomorphic basaloid cells forming nests, cords, and frequent cribriform patterns. Squamous dysplasia of surface mucosa, focal squamous differentiation within invasive basaloid squamous cell carcinoma, or foci of conventional squamous cell carcinoma were present, alone or in combination. All studied neoplasms were immunohistochemically positive for keratins with the 34 beta E12 antibody. Approximately 80% were immunoreactive using AE1/AE3 or CAM 5.2. Epithelial membrane antigen, carcinoembryonic antigen, and S100 protein were found in 83%, 53%, and 39%, respectively, of the cases. Diffuse, weak immunoreactivity for neuron-specific enolase was seen in 75% of tumors. Synaptophysin, chromogranin, muscle-specific actin, and glial fibrillary acidic protein were absent. Basaloid squamous cell carcinoma has been confused with adenoid cystic carcinoma and small cell undifferentiated carcinoma, but is usually distinguishable in routine hematoxylin and eosin-stained sections, or, in rare problematic cases, with the aid of immunohistochemical studies. Distinction is warranted because the biologic behavior of basaloid squamous cell carcinoma differs from that of both of these lesions.


Intermediate filamentous proteins in adult granulosa cell tumors. An immunohistochemical study of 25 cases. Adult granulosa cell tumors (AGCTs) are classified as sex cord-stromal tumors of the ovary. However, they may be confused with other primary ovarian neoplasms. Intermediate filaments, specifically vimentin and cytokeratins, have been identified in AGCTs by immunohistochemistry performed on frozen and formalin-fixed, paraffin-embedded tissue and two-dimensional electrophoresis. Recently, however, immunohistochemical demonstration of cytokeratin has been used as supporting evidence of epithelial rather than sex cord-stromal differentiation in ovarian neoplasia. To investigate further intermediate filamentous proteins in AGCTs, 25 such tumors were studied by immunohistochemistry in formalin-fixed, paraffin-embedded sections. Cytoplasmic staining was observed, frequently in a distinct punctate, paranuclear pattern, in 14 of 25, 14 of 25, and seven of 17 tumors using monoclonal antibodies AE1/AE3, CAM 5.2, and 35BH11, respectively, which share the ability to detect low molecular weight cytokeratins. Staining for cytokeratin was not seen in any of the 17 tumors studied using the antibody 34BE12. Twenty-three of 25 tumors showed strong positivity for vimentin, characteristically seen as globoid paranuclear staining. Nine of 25 tumors contained desmin, which was restricted to the intermixed spindle cell, cortical type stromal component of the tumors. These patterns of immunoreactivity for intermediate filaments, particularly cytokeratins, are different than in common epithelial tumors of the ovary and may be useful in the differential diagnosis of ovarian neoplasia. Moreover, the immunohistochemical detection of cytokeratins should not be used as a criterion for excluding AGCT from the differential diagnosis of an ovarian neoplasm.


Malignant pleural mesothelioma producing human chorionic gonadotropin. Report of two cases. Two cases of malignant pleural tumor producing human chorionic gonadotropin, one confirmed at surgery and autopsy and the other by biopsy, are reported. Both of the patients had bilateral gynecomastia with high levels of serum human chorionic gonadotropin. The first patient underwent panpleuropneumonectomy because of diffuse malignant pleural tumor, but died five months later due to recurrent disease. The histological diagnosis was diffuse, malignant, monophasic mesothelioma of the epithelial type. Immunostaining for alpha-, beta-human chorionic gonadotropin and human placental lactogen was positive in syncytiotrophoblast-like cells. The second patient had diffuse pleural tumor with massive effusion. A pleural biopsy specimen showed diffuse proliferation of epithelioid large cells. Immunostaining for alpha, beta-human chorionic gonadotropin and human placental lactogen was positive in mono- and multinucleated bizarre giant cells mimicking trophoblasts. A diagnosis of malignant mesothelioma with trophoblastic differentiation seemed most likely in both cases in view of the clinical and pathological findings. In both cases, results of mucin histochemistry and various immunohistochemical stains for antigens or with antibodies were consistent with diagnosis of mesothelioma except in a few cells in the choriocarcinomatous portion. This may be the first report describing human chorionic gonadotropin-producing malignant mesotheliomas of the pleura.


Neoplastic epithelial cells in a subset of human thymomas express the B cell-associated CD20 antigen. A series of 36 human thymomas have been immunohistochemically analyzed using a panel of antibodies recognizing B-cell markers including CD20. Most thymomas exhibiting the cortical pattern, according to the criteria of Marino and Muller-Hermelink, were characterized by areas of medullary differentiation containing variable numbers of CD20+ B lymphocytes, thus mimicking the medulla of normal thymus. On the other hand, B cells were absent or rare in thymomas recognized as mixed using the same morphological criteria. Surprisingly, we observed in most mixed thymomas variable numbers of CD20+ spindle cells, characterized by long slender processes. Using double-marker analysis we could demonstrate the epithelial nature of these cells (expression of keratin and lack of lymphoid and B-cell-related markers). The immunoreactivity of thymoma epithelial cells with L26, an antibody widely used in the characterization of B-cell lymphomas, can represent a drawback of practical relevance in the differential diagnosis of mediastinal tumors.


Desmoplastic primitive neuroectodermal tumor with divergent differentiation. Broadening the spectrum of desmoplastic infantile neuroepithelial tumors. We report an unusual large, multicystic, posterior fossa neuroepithelial neoplasm involving the cerebellum, brain-stem, and quadrigeminal cistern of a 9-month-old girl. The neoplasm consisted of variably sized, sharply demarcated nests of small cells with a high nuclear-cytoplasmic ratio and moderately basophilic nuclei, embedded in a desmoplastic, immature-appearing, mesenchymal stroma. The nests contained mitoses but none were seen in the stroma. Glial fibrillary acidic protein (GFAP), neurofilament protein, synaptophysin, and cytokeratin (AE-1) were expressed in the nests. Mesenchymal cells were negative for neural markers but positive for vimentin and desmin. The neoplasm was interpreted as a mixed mesenchymal and primitive neuroectodermal tumor (PNET) with histologic features reminiscent of a recently described intraabdominal desmoplastic small cell tumor. The tumor responded poorly to chemotherapy and a second operation was performed 1 year later. The second specimen bore no resemblance to the original and consisted of epithelial-like nests and clusters of neoplastic cells frequently interrupted by sinusoidal vessels. Tumor cells had medium-sized vesicular nuclei with small nucleoli, and a granular cytoplasm. Occasional less cellular islands of neuropil-like tissue contained larger cells having eccentric, vesicular nuclei with prominent nucleoli and abundant pink cytoplasm. Mitoses were not conspicuous. Many cells expressed synaptophysin, neurofilament protein, and GFAP. Neurofilament protein was strongly positive in the larger, neuron-like cells and synaptophysin stained the neuropil-like areas strongly but was less prominent in the neuronal perikarya. Unexpectedly, the neuropil-like areas expressed epithelial membrane antigen, whereas the neuronal cells were negative for chromogranin A. The peculiar histologic picture, combination of phenotypic markers, and remarkable biologic behavior of this unusual tumor defies classification according to existing nomenclature and exemplifies the broad range of phenotypes expressed by primitive neuro-epithelial neoplasms.


Acinar cell carcinoma of the pancreas. A clinicopathologic study of 28 cases. We have examined the microscopic appearance, immunohistochemical staining properties, and clinical behavior of 28 cases of acinar cell carcinoma of the pancreas. Two of the tumors occurred in children. The adult patients ranged in age from 40 to 81 years (mean, 62 years). Males greatly outnumbered females, and most of the patients were white. Presenting symptoms were nonspecific, and jaundice was infrequent. The frequently reported complications from increased serum lipase levels (i.e., arthralgias and subcutaneous fat necrosis) were present in only 16% of the patients. Grossly, the tumors were relatively circumscribed and fleshy, averaging 10.8 cm, with occasionally extensive hemorrhage and necrosis. Microscopically, the tumors were very cellular and characteristically lacked a desmoplastic stroma. Acinar, solid, trabecular, and glandular patterns of growth were identified; individual tumors were usually mixed. Nuclei were round to oval, with minimal pleomorphism and single prominent nucleoli. Mitotic activity was variable. In general the cytoplasm was moderately abundant, eosinophilic, and granular, but many of the solid tumors had cells with scanty cytoplasm. Characteristic periodic acid-Schiff-positive, diastase-resistant cytoplasmic granules were demonstrated in greater than 90% of the cases, and the butyrate esterase histochemical stain for lipase activity was positive in 73%. Immunohistochemically, there was positivity for trypsin in 100% of the cases, for lipase in 77%, for chymotrypsin in 38%, and for amylase in 31%. A minor endocrine component was recognized with antibodies against chromogranin or islet cell hormones in 42% of the tumors. Ultrastructurally, exocrine secretory features were present, with polarized cells showing microvillilined lumina, abundant rough endoplasmic reticulum, and 125-1,000-nm zymogen-like granules. In addition, many cases showed pleomorphic electron-dense granules measuring up to 3,500 nm and containing fibrillary internal structures. Follow-up information was available in 88% of the cases. Half of the patients had metastatic disease at presentation and an additional 23% subsequently developed metastases, which were usually restricted to the regional lymph nodes and liver. The mean survival for all cases was 18 months, with 1- and 3-year survivals of 57 and 26%, respectively. Patients presenting before age 60 years survived nearly twice as long as older patients did. Stage also influenced prognosis, whereas the histologic subtype of the tumors and the location within the pancreas correlated only weakly with survival.


Metastasizing mixed tumor of salivary glands. A clinicopathologic and flow cytometric analysis. Among salivary gland neoplasms are a group of rare tumors that are histologically identical to benign mixed tumors that inexplicably metastasize; they have been called metastasizing mixed tumor (MZMT) of salivary glands. We report the clinicopathologic features and flow cytometric findings for 11 cases of MZMT. At the time of discovery of metastatic disease, the patients, six women and five men, ranged in age from 20 to 83 years. Primary sites of involvement included the parotid gland (eight cases), submandibular gland (two cases), and the nasal septum (one case). With one exception, all the patients had at least a single recurrences of their primary mixed tumor, but two or more recurrences were the norm before development of metastatic foci. The metastases were discovered from six to 52 years following the occurrence of the primary tumor. Metastatic deposits were identified in bone, lung, regional lymph nodes, skin, kidney, retroperitoneum, oral cavity, pharynx, calvarium, and central nervous system. The metastases either occurred simultaneously with an episode of recurrent mixed tumor (n = 5) or from 5 to 29 years after a recurrence (n = 6). The treatment of the primary, recurrent, and metastatic neoplasms was surgical excision. Follow-up, ranging from 8 months to 16 years following the diagnosis of MZMT, revealed seven patients to be alive without disease (64%) and two dead of causes unrelated to metastatic disease (18%). Two patients (18%) died as a direct result of metastatic tumor at 3 and 2 years after metastasis of their mixed tumors. Flow cytometric analysis revealed a diploid DNA cell population in the primary and/or metastatic tumors in nine cases. Aneuploid DNA cell content was identified in two of the cases. DNA ploidy levels and cell proliferation rates were compared with those of conventional benign mixed tumors and also with malignant mixed tumors. Retrospective analysis of histologic parameters (mitotic rate, cellular pleomorphism, infiltrative growth, vascular or lymphatic invasion) and flow cytometric analysis failed to identify criteria to predict the development of metastasis in these neoplasms.


Chronic lymphocytic leukemia/small lymphocytic lymphoma with Reed-Sternberg-like cells and possible transformation to Hodgkin's disease. Mediation by Epstein-Barr virus. The pathogenesis of Reed-Sternberg cells and variants (RS-H cells) found in rare cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is unknown. We studied 13 such cases by immunohistochemistry and in situ hybridization for identification of Epstein-Barr virus (EBV) RNA. The RS-H cells in five cases expressed the B-lineage marker CD20 and were negative for CD15. In two cases, the RS-H cells showed expression of both CD20 and CD15, whereas in another six cases, the cells were positive for CD15 but negative for CD20. Three of the cases expressing CD15 showed subsequent evidence of disseminated Hodgkin's disease. Regardless of the phenotype or clinical behavior, the RS-H cells in 12 of 13 cases were found to contain EBV RNA by in situ hybridization, but the surrounding neoplastic lymphocytes were invariably negative for EBV RNA. It is suggested that EBV has an important role in the pathogenesis of the RS-H cells in these rare cases.


Adenomyoepithelioma of the breast. A spectrum of biologic behavior. Adenomyoepitheliomas of the breast have been considered to have limited metastatic potential; axillary node metastasis has been reported, but there has been no report of distant metastasis. We report six cases, including two malignant adenomyoepitheliomas, one of which metastasized to the lung and brain. Patient age ranged from 26 to 63 years (mean 46). Primary tumors were solitary and measured 0.9-3.5 cm (mean 1.7). Five of six tumors presented as palpable masses. Two patients treated by local resection have no evidence of disease at 5 and 18 months' follow-up. Two patients treated by local resection had recurrences, one at 48 the other at 60 months. The fifth patient had a spindle-cell type adenomyoepithelioma diagnosed as malignant because of high mitotic rate and cytologic atypicality of the myoepithelial component. This patient was treated by mastectomy and has no evidence of disease at 18 months. The sixth patient, initially treated by local excision, had six local recurrences over 52 months treated by reexcisions, mastectomies, and radiation. A lung metastasis was resected at 54 months and brain metastases were identified at 60 months with death occurring at 64 months. Both malignant adenomyoepitheliomas had high mitotic rates [11-14/10 high-power fields (HPF)] diffusely throughout the tumors and foci of cytologically malignant cells. The malignant adenomyoepithelioma that metastasized had an infiltrative growth pattern that increased with successive local recurrences. The four other tumors had only isolated areas of mitotic activity (maximum 1-9/10 HPF) and minimal cytologic atypia. Immunohistochemistry performed on five of six cases confirmed dual epithelial/myoepithelial cell populations in all tumors examined, including the metastasis. Electron microscopic examination of the malignant adenomyoepithelioma that metastasized also confirmed dual epithelial/myoepithelial cell populations in a local recurrence and the lung metastasis. We conclude that there is a spectrum of behavior for breast adenomyoepitheliomas with potential for local recurrence and, rarely, distant metastasis.


Lymphoepitheliomalike carcinoma of the skin. A case report with immunophenotypic analysis and in situ hybridization for Epstein-Barr viral genome. Lymphoepithelioma is a malignant epithelial neoplasm of the nasopharynx. Similar malignancies--lymphoepitheliomalike carcinomas--of salivary glands, thymus, larynx, lung, stomach, and uterus have been described. We present here a case of lymphoepitheliomalike carcinoma of the skin in an 84-year-old woman. Histologically this neoplasm presented as a fairly discrete dermal aggregate of syncytial nests of epithelioid-appearing cells that displayed no squamous or glandular differentiation, surrounded by a dense lymphocytic infiltrate. Results of immunophenotypic studies showed expression of high molecular weight cytokeratins and increased density of dermal dendrocytes within and adjacent to the tumor. No Epstein-Barr viral genomic sequences were detected by in situ hybridization, which suggests that cutaneous neoplasms may have different etiologic agents compared with similar tumors, found to be associated with this DNA-containing virus, arising in extracutaneous sites. The combined light microscopic and immunohistochemical assessment of this rate cutaneous neoplasm permits distinction of lymphoepitheliomalike carcinoma from benign/malignant lymphoproliferative disorders or neuroendocrine carcinomas.


Malignant eccrine spiradenoma. A clinicopathologic study. Malignant eccrine spiradenomas (MES) are exceedingly rare and their immunohistochemical and ultrastructural features have not been fully characterized. We studied two cases, one of them immunohistochemically and electron microscopically. Patient 1 had a 25-year history of multiple exophytic tumors involving the scalp, the skin of the face, and the torso. Of the lesions removed, ten were spiradenomas, two with malignant changes, and three were cylindromas. The malignant areas showed loss of tubular and nesting patterns, lack of two cell populations, and contained anaplastic cells with high mitotic rate. The immunohistochemical findings were consistent with eccrine differentiation. Patient 2 had a cystlike mass of long duration in the right groin. Histologically, the mass consisted of nodules of benign eccrine spiradenomas adjacent to a ductal-cystic mass lined by anaplastic cells, but areas of squamous and glandular differentiation were also present. (a) Case 1 is probably the first reported MES associated with multiple spiradenomas and cylindromas. (b) Cytodifferentiation in MES is variable, sometimes with almost complete loss of eccrine differentiation. (c) Identification of adjacent spiradenomas may be required for definite diagnosis of MES. (d) Clinical history of longstanding lesions with recent fast growth warrants tissue diagnosis.


Immunohistochemical stains in extramammary Paget's disease. The histologic and immunohistochemical characteristics of 49 skin biopsy specimens from 49 patients with extramammary Paget's disease were studied. Patients with extramammary Paget's disease with and without underlying malignant disease were identified. Associated malignant lesions, present in 16 patients (33%), were transitional cell carcinoma of the bladder (n = 8), adenocarcinoma underlying the skin (n = 3), adenocarcinoma of the anus (n = 1), adenocarcinoma of the vulva (n = 1), apocrine carcinoma (n = 1), prostate carcinoma (n = 1), and carcinoma metastatic to the lung (n = 1). The main histologic feature was the presence of Paget's cells, predominantly at the base of the epidermis. In 6% of the cases, well-defined nests of large Paget's cells mimicked melanocytic nests. Carcinoembryonic antigen and Cam 5.2 (a monoclonal antibody that stains 40-kDa, 45-kDa, and 52.5-kDa low molecular weight keratins) were localized to the Paget's cells in 42 of 45 (93%) and 29 of 41 cases (71%), respectively. Forty-four of 46 lesions (96%) were mucin positive, as determined by Hale's colloidal iron stain. Absence of staining for colloidal iron and carcinoembryonic antigen occurred somewhat more frequently in patients with underlying malignant disease than in patients without tumors (13% vs. 0% mucin negative and 13% vs. 3% carcinoembryonic antigen negative, respectively). Although immunohistochemical staining for low molecular weight keratin may be used to confirm the diagnosis of extramammary Paget's disease, Cam 5.2 is not as sensitive as the colloidal iron or carcinoembryonic antigen stain.


Microplate reader-based quantitation of collagens. By using a picrosirius dye, sensitive and specific staining of collagens plated in microtiter wells was achieved. The range of detection was from 0.5 to 20 micrograms. Human collagen types I, III, IV, and V were tested and able to be detected by the method. The dye did not bind to acetylcholinesterase or elastin. It did bind to C1q to some extent but this is not surprising since the molecule contains some triple helical collagen-like structures. A comparison performed between this assay and a colorimetric assay for hydroxyproline using tissue culture supernatants gave similar results for both samples. Due to its simplicity and sensitivity this assay will be most useful in laboratories where large numbers of samples must be screened for collagen production.


A spectrophotometric assay of Zn(2+)-glycerophosphorylcholine phosphocholine phosphodiesterase using p-nitrophenylphosphorylcholine. A direct spectrophotometric assay for the glycerophosphorylcholine phosphocholine phosphodiesterase requiring zinc ions for activity is described. This assay is based on the continuous measurement of p-nitrophenol produced from the enzymatic hydrolysis of p-nitrophenylphosphorylcholine. The assay method, which showed a good linearity with time and amount of protein, was found to be rapid, simple, and, at the same time, accurate and sensitive enough to allow the quantitation of nanomolar amounts of product. With an alkaline buffer containing Triton X-100, the Zn(2+)-glycerophosphorylcholine phosphocholine phosphodiesterase activity in the tissue homogenate can be directly and selectively measured by this technique. The specific activity of the phosphodiesterase in brain and kidney was determined to be 80 and 6.5 nmol/h mg protein, respectively, and much lower activity was present in other tissues.


Rapid analysis of mitotic histone H1 phosphorylation by cationic disc electrophoresis at neutral pH in minigels. A new method is described for analysis of histone H1 and other basic proteins by cationic disc electrophoresis in polyacrylamide gels at neutral pH. The multiphasic buffer (disc) system uses Na+ as leading ion, L-histidine as trailing ion, and Hepes as buffering counterion. These "Hepes/histidine gels" have three advantages over conventional acid-urea gels for studies of H1 phosphorylation and dephosphorylation: speed, convenience, and the need for only small amounts of cells or chromatin. Core histones and their acetylated forms can also be separated in gels containing 0.4% Triton X-100. The difference in electrophoretic mobility between mitotic (superphosphorylated) and interphase H1 from HeLa cells is approximately twice as great at neutral pH as at pH 4.5, making it possible to separate these two H1 forms rapidly and easily in Hepes/histidine "minigels" only 5-cm long. Total histones can be rapidly prepared by simply neutralizing 0.2 N HCl extracts, and the entire analysis, from harvesting cells to destaining gels, can be carried out in 1 day. The stacking effect of the disc system produces sharp bands and high resolution even with relatively dilute samples.


Incorporation of a bacterial membrane-bound hydrogenase into proteoliposomes. Membrane-bound nickel-iron hydrogenases from diverse genera of bacteria have been previously characterized and they are closely related. We report the reconstitution of purified Bradyrhizobium japonicum hydrogenase into proteoliposomes by a detergent dialysis method followed by two or three cycles of freeze-thaw. Sedimentation experiments revealed that more than 60% of the H2-uptake activity was particulate when reconstituted into Escherichia coli phospholipids. Sucrose-gradient centrifugation separated hydrogenase activity into two peaks, the less dense of which was phospholipid-associated and turbid, thereby showing successful incorporation. Purified enzyme did not bind to performed phospholipid vesicles, and 1.0 M NaCl failed to remove incorporated hydrogenase. The optimal micellar detergent:phospholipid ratio (rho) value for hydrogenase incorporation was 2.0. Proteoliposomes containing acidic phospholipids were the most effective for incorporation as well as for activity. The artificial electron acceptor specificity was similar for proteoliposomes and for H2-oxidizing membranes from B. japonicum. Proteoliposomes formed under optimal conditions had a broad size distribution centered around 400 nm diameter. Hydrogenase activity in proteoliposomes was partially protected from inactivation by the protein modification reagent diazobenzene sulfonate (DABS) (inactivation t1/2 = 30 min), whereas DABS rapidly inactivated the purified enzyme (t1/2 = 4 min). The latter result indicates protection of a catalytically important site by the phospholipid bilayer. This experimental system should be useful in addressing questions regarding the in vivo situation of bacterial membrane-bound hydrogenases.


A rapid and versatile method for cloning viroids or other circular plant pathogenic RNAs. We surveyed the occurrence of unique restriction sites on the cDNAs of viroids, virusoids, and plant viral satellite RNAs that have a circular RNA as an intermediate of replication and found that four such sites would linearize their circular cDNAs. A rapid and simple method was then developed for cloning a naturally occurring viroid from Nematanthus wettsteinii plants. First-strand cDNA was synthesized using random hexanucleotide DNA primers and M-MuLV reverse transcriptase (Superscript RT). Second-strand DNA was synthesized by employing the replacement synthesis method using Escherichia coli RNase H, E. coli DNA polymerase I, E. coli DNA ligase, and beta-NAD+. The circular double-stranded DNA was analyzed for the presence of commonly available, unique restriction sites and subsequently linearized with a selected restriction enzyme. The linear cDNA was ligated to dephosphorylated plasmid vector pGEM 3Z f(+) and cloned in E. coli strain DH5 alpha. This cDNA cloning procedure is suitable for cloning sequence variants of well-characterized viroids, virusoids, certain plant viral satellite RNAs, and new such pathogens of unknown sequence.


A semiautomated, 96-well plate assay for collagen synthesis. We have established a rapid method for the measurement of collagen synthesis in large numbers of cell cultures. 3H-labeled collagen in microwell cultures was salt precipitated, harvested, and washed using a commercially available cell harvester and the filtered collagen was directly counted. The cell number could then be assessed either by methylene blue staining or by dissolving the cells in NaOH and estimating the protein content with bicinchoninic acid. In all of these procedures, the samples remain in microtiter plates, thus ensuring that minimal amounts of reagents are used and minimizing the amount of manipulation necessary. The intergroup variability is 3 to 9% and the recovery of 3H-labeled collagen is greater than 90%. Using this method, large numbers of samples can be assessed for collagen synthesis quickly, conveniently, and for minimal cost.