Effects of free and bound insulin-like growth factors on proteoglycan metabolism in articular cartilage explants.

This article describes the effects of bound forms of insulin-like growth factors (IGFs) on proteoglycan metabolism by bovine articular cartilage in explant culture. When these growth factors were added to articular cartilage explants complexed with their native serum binding proteins (BPs), both IGF-I-BP complex and IGF-II-BP complex stimulated proteoglycan synthesis to different degrees over a 3-day period. When added to the medium of cultures of articular cartilage over 5 days, IGF-II-BP complex induced high rates of synthesis and low rates of catabolism of proteoglycans, giving rise to tissue levels of proteoglycan similar to those observed in fresh tissue. When articular cartilage was maintained in culture with the same concentration of IGF-I-BP complex, tissue levels of proteoglycans fell over the culture period because of lower rates of proteoglycan synthesis. Analysis of the proteoglycans synthesized by articular cartilage in the presence of free or bound IGF-I or IGF-II showed that these growth factors stimulated the rate of synthesis of the large proteoglycan species present in cartilage but did not affect the synthesis of the small proteoglycans.


S-100 protein immunostaining identifies cells expressing a chondrocytic phenotype during articular cartilage repair.

The healing of articular surface defects has been studied with conventional histology, which relies on the staining of the extracellular matrix to identify the phenotype of the cells present. A chondrospecific cellular marker would be useful. S-100 protein has been found in all chondroid tissues studied, and we evaluated its usefulness in the study of articular cartilage repair. Full-thickness rabbit femoral condylar defects were made, and the specimens were studied at serial time intervals. S-100 protein staining positively showed chondroid cells in the 7- and 14-day specimens, which were not identifiable by conventional techniques. At 30 and 60 days, an S-100 positive band of cells separated a deep safranin-O positive hypertrophic layer from a fibrocellular surface layer. At 120 days, the presence of S-100 protein identified cells with chondrogenic potential, and the lack of S-100 protein in other cells embedded in conventionally stained matrix suggested that these cells were no longer of a chondroid phenotype. The presence of S-100 protein-identified chondroid cells early in the repair process when the cells had not begun to synthesize conventionally stainable matrix and the lack of S-100 protein in cells late in the repair positively identified a phenotypic change earlier than conventional histology.


Rabbit knee immobilization: bone remodeling precedes cartilage degradation.

This study analyzed processes underlying osteoporosis and osteoarthrosis after short-term immobilization of the right hind limb of postadolescent (2.8 kg) and mature (4.0 kg) rabbits. After 3 weeks, the lateral posterior aspect of the lateral tibial plateau and the lateral femoral condyle of the immobilized limb exhibited prominent subchondral vascular eruptions. Femoral metaphyseal bone density decreased 27 and 18% in the immobilized limbs of postadolescent and mature rabbits, respectively. Calcein green fluorescence increased 1.9-fold (p less than 0.001) in the metaphyseal trabeculae of immobilized femurs. With immobilization, sulfate incorporation into femoral cartilage glycosaminoglycan increased, although total cartilage glycosaminoglycan and hydroxyproline levels were unchanged. Thymidine incorporation into DNA increased four- to fivefold in tibial and femoral cartilage of the immobilized limb. In this study, bone loss and remodeling preceded erosive cartilage degradation.


The Denver II: a major revision and restandardization of the Denver Developmental Screening Test.

Since the Denver Developmental Screening Test was first published 23 years ago, it has been utilized worldwide and restandardized in more than a dozen countries. Concerns raised through the years by test users about specific items and features of the Denver Developmental Screening Test, coupled with a need for more current norms, have prompted a major revision and restandardization of the test. For the revision, 336 potential items were administered to more than 2000 children. The average number of times each item was administered was 540. Using regression analysis, composite norms for the total sample and norms for subgroups (based on gender, ethnicity, maternal education, and place of residence), were used to determine new age norms. The final selection of the 125 Denver II items was based on the following criteria: ease of administration and scoring, item appeal to child and examiner, item test-retest and inter-rater reliability, minimal "refusal" scores, minimal "no opportunity" scores, minimal subgroup differences, and a smooth step-like progression of ages at which 90% of children could perform the tasks. The major differences between the Denver II and the Denver Developmental Screening Test are: 1) an 86% increase in language items; 2) two articulation items; 3) a new age scale; 4) a new category of item interpretation to identify milder delays; 6) a behavior rating scale; and 7) new training materials.


Participation by pediatricians in early intervention: impetus from Public Law 99-457.

Part H of the Individuals with Disabilities Education Act (originally enacted as Public Law 99-457) requires that participating states phase in a system of early intervention services by 1993. By recognizing the importance of good health in the development of infants and toddlers, Congress acknowledged the key role of medical care providers in a comprehensive program for young children with or at risk for developmental delay or dysfunction. National and state surveys of pediatricians suggest limited but growing awareness of this legislation and uncertainty about how they might participate effectively. A chief concern relates to mechanisms of payment for developmental screening and assessment as well as time-demands for participation in interdisciplinary team activities. The American Academy of Pediatrics and its state chapters are responding to requests for information with educational seminars and print materials. Pediatricians can enhance the quality of community support services for children with special needs by participating in planning efforts and by coordinating health care with other aspects of early intervention. Other professionals and parents are looking to pediatricians for leadership and willing participation in the implementation of PL 99-457.


Differential effects of dichlorodiphenyltrichloroethane analogs, chlordecone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin on establishment of pregnancy in the hypophysectomized rat.

Many of the organochlorine pesticides have been shown to elicit estrogenic responses in laboratory animals. Two estrogenic actions, initiation of implantation and maintenance of pregnancy, were examined in progesterone-primed, delayed-implanting, hypophysectomized rats exposed to several polychlorinated hydrocarbons. The insecticide P,P'-dichlorodiphenyltrichloroethane (DDT) was nearly devoid of estrogenic activity for initiating implantation, as was a dichloro analog, 1,1-dichloro-2-[p-chlorophenyl],2-[o-chlorophenyl]ethane (O,P'-DDD), but another such analog, 1,1-dichloro-2-(p-chlorophenyl),2-(o-chlorophenyl)ethylene (O,P'-DDE), was nearly as estrogenic as the O,P'-DDT isomer of DDT and the methoxylated analog methoxychlor. The latter three compounds not only initiated implantation, but maintained pregnancy when given in large (200 mg/kg) and repeated doses. Another insecticide, chlordecone (Kepone) was more estrogenic than any of the DDT analogs and maintained pregnancy with a single dose of 50 mg/kg. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a toxic contaminant of herbicide production, did not induce implantation at a dose of 125 micrograms/kg, but inhibited the implantation initiated by estrone in 35% of the animals. The mechanism of this antiestrogenicity is unknown but most probably does not involve direct action via the classical estrogen receptor. The possible interference with the normal blastocyst-uterine interactions of these polychlorinated xenobiotics may be an important factor in their being considered reproductive toxins.


Effectiveness of India ink as a long-term colonic mucosal marker.

We prospectively studied the use of India ink as a long-term or "permanent" mucosal marker as part of a study investigating the natural history of diminutive distal colorectal polyps. Twenty-six patients had 32 India ink tatoos implanted. The tatoo sites of the 19 patients who were followed at least 6 months continued to display intensely stained mucosa at the original sites. No side effects or complications were encountered. India ink appears to be a safe and effective long-term marker for colonic mucosal lesions.


Morphometric comparisons of optic nerve axon loss in acquired immunodeficiency syndrome.

Axonal degeneration and diminution of the axonal population in the optic nerve have been documented in aging and in various neuro-ophthalmic conditions. We applied morphometric techniques to the postmortem examination of optic nerves obtained from patients with acquired immunodeficiency syndrome. Twelve optic nerves (eight from patients with AIDS and four from age-matched control eyes) were stained with paraphenylenediamine and morphometrically analyzed with a computer-assisted image and measurement system. Degeneration was often severe and was scattered throughout all of the AIDS-affected optic nerves. In the AIDS-affected optic nerves, the mean axonal population was markedly lower than the mean obtained from normal optic nerves (880,000 vs 1,507,000). Despite the approximate 40% loss of axons, mean axonal diameters were not markedly different, suggesting that no particular class of axon was especially susceptible to AIDS-associated degeneration. The extent and pattern of axonal loss in optic nerves of patients with AIDS suggest that the changes may not only be secondary to damage at the retina, but may reflect an AIDS-associated primary optic neuropathy.


Crystalloids in angiomyolipoma. 1. A previously unnoticed phenomenon of renal angiomyolipoma occurring at a high frequency.

We present a description of unique crystalloids in renal angiomyolipoma that have not previously been reported. The crystalloids cannot be identified by hematoxylin-and-eosin staining. Detailed observation after diastase treatment followed by PAS staining revealed needle- and rod-like crystalloids, which were clearly seen even by light microscopy, in 11 of 17 patients. Their appearance was characterized by the following phenomena: (a) They appeared mainly in large epithelioid smooth-muscle cells; (b) they appeared at a relatively high frequency at sites where smooth-muscle cells showed diffuse proliferation and where a hemangiopericytic pattern was observed; (c) they were often detected easily even at a site with a sarcomatous appearance; and (d) PAS-positive, diastase-resistant granules were often observed by light microscopy in the vicinity of crystalloids in all 17 patients. Electron-microscopic observation of one patient also revealed characteristic crystalloids. Prior to our study, only one patient had been reported to show crystalloids by electron microscopy, and the crystalloids were interpreted as renin. However, our study used Bowie's staining and immunohistochemistry to prove they were not renin. The nature of the crystalloids still needs to be elucidated. The fact that they closely resemble structures seen in alveolar soft part sarcoma provides one clue to their identification.


Bile duct adenomas with endocrine component. Immunohistochemical study and comparison with conventional bile duct adenomas.

Bile duct adenomas are small nodules that are usually found incidentally on the liver surface at abdominal surgery or autopsy. We recently analyzed two such lesions that, in addition to the typical small caliber ducts, contained periductular nests and clusters of uniform round cells, suggestive of endocrine cell proliferation. Follow-up of these patients did not show endocrine tumors elsewhere. The lesions were studied by immunohistochemistry (avidin-biotin-peroxidase technique) and compared with conventional bile duct adenomas (seven cases). The results showed these cells to decorate with several endocrine markers, namely, neuron-specific enolase, chromogranin, synaptophysin, and Leu-7. Endocrine markers were not seen in the cells of conventional bile duct adenomas. Epithelial markers, that is, cytokeratin (CAM 5.2 antibody) and epithelial membrane antigen, were expressed by the cells composing both conventional bile duct adenomas and those with endocrine-like cells, although with less intensity in the endocrine cell clusters. We suggest that some bile duct adenomas contain endocrine cell proliferations that morphologically may resemble a small carcinoid tumor or the so-called pulmonary tumorlet. Neurosecretory granules have previously been identified in some cholangiocarcinomas and in bile duct proliferation associated with cholestasis. The endocrine clusters in biliary adenomas may constitute a diagnostic pitfall and must be separated from metastases of carcinoids or islet cell tumors.


Routine diagnosis of mammary Paget's disease. A modern approach.

This study compares the diagnostic reliability of conventional mucin histochemistry and immunocytochemical techniques in distinguishing mammary Paget's disease from superficial spreading malignant melanoma and primary intraepidermal carcinoma. Formalin-fixed, paraffin-embedded archival tissue was used and comprised 13 cases of mammary Paget's disease, five cases of superficial spreading melanoma, and six cases of intraepidermal carcinoma. Sections from each case were stained for the presence of mucin using diastase periodic-acid-Schiff (d-PAS) with and without an alcian blue counterstain as well as immunocytochemistry for cytokeratin (CAM 5.2), epithelial membrane antigen (NCRC-11) and c-erb B-2 (21N). Mucin staining in intraepidermal carcinoma and malignant melanoma was consistently negative. Diastase-resistant PAS positivity was seen in six of 13 cases of mammary Paget's disease and eight of 13 cases using an alcian blue counterstain. NCRC-11 showed positive immunoreactivity in four of six cases of intraepidermal carcinoma, one in five cases of melanoma, and five of 13 cases of mammary Paget's disease. Positive immunoreactivity using CAM 5.2 and 21N was seen in all cases of mammary Paget's disease, with consistent negative immunoreactivity in the other tumor types. We conclude that CAM 5.2 and 21N should be used in the investigation of mammary Paget's disease in preference to conventional mucin stains.


Paneth cell-like change of the prostate gland. A histological, immunohistochemical, and electron microscopic study.

Paneth cell-like change (PCLC) of the prostatic glandular epithelium was focally observed in one case of normal glandular epithelium, two cases of glandular and stromal hyperplasia, one case of prostatic intraepithelial neoplasia, and four cases of prostatic adenocarcinoma. The distinctive cells were characterized by bright, eosinophilic cytoplasmic granules on routine hematoxylin and eosin-stained material. The cytoplasmic granules in the benign prostatic epithelium were periodate-Schiff's procedure (PAS)-positive and diastase resistant and immunohistochemically negative for lysozyme, neuron-specific enolase, chromogranin, and serotonin. The eosinophilic granules in the prostatic intraepithelial neoplasia and adenocarcinoma cases were immunohistochemically positive for chromogranin, serotonin, and neuron-specific enolase, and negative for lysozyme. By electron microscopy the eosinophilic granules represented exocrine-like or lysosomal-like vesicles in the benign epithelium and neuro-endocrine granules in the malignant epithelium. The lesion represents a prostatic epithelial PCLC rather than a Paneth cell metaplasia. PCLC is the common histological manifestation of two different phenomena: (a) a PAS-positive and diastase-resistant eosinophilic cytoplasmic granular change in benign prostatic epithelium, and (b) endocrine differentiation with neuroendocrine granules in dysplastic and malignant prostatic epithelia. The importance of recognizing PCLC lies in its differentiation from other possible prostatic cytoplasmic inclusions.


A 30-s PAS stain for frozen sections.

A modified periodate-Schiff's procedure technique for application in frozen section diagnosis is described. This simplified reaction performed using a microwave-oven procedure takes only 30 s and is useful in demonstrating mucin as well as mucin-containing cells. We found that this method facilitates frozen-section diagnosis.


Primary lung cancer producing alpha-fetoprotein.

Reports of alpha-fetoprotein-producing lung tumors are rare. Only 24 such patients with these tumors have been previously studied. We report the case of a patient with a large cell carcinoma of the lung and a serum alpha-fetoprotein level of 9,300 ng/mL, with no evidence of hepatic or other systemic abnormalities. Serum levels of alpha-fetoprotein returned to normal postoperatively.


Cerebrospinal fluid neurochemistry in children and adolescents with obsessive-compulsive disorder.

Cerebrospinal fluid hormones, monoaminergic metabolites, and dynorphin A (1-8 sequence) were examined in 43 children with severe, primary obsessive-compulsive disorder. Cerebrospinal fluid levels of 5-hydroxyindoleacetic acid were positively correlated with one of eight obsessive-compulsive disorder severity ratings and three of seven measures of improvement following 5 weeks of treatment with clomipramine hydrochloride. Arginine vasopressin concentration was significantly and negatively correlated with several ratings of obsessive-compulsive disorder symptom severity, while oxytocin concentration was positively correlated with depressive symptoms. The ratio of arginine vasopressin to oxytocin was also negatively correlated with obsessive-compulsive disorder and depressive symptoms. Comorbid affective disorder was associated with decreased arginine vasopressin concentrations, while concomitant anxiety disorder was associated with increased oxytocin. Dynorphin A (1-8 sequence), homovanillic acid, corticotropin, 3-methoxy-4-hydroxyphenylglycol, and corticotropin releasing hormone were not significantly related to obsessive-compulsive disorder symptoms. These results seem to indicate that arginine vasopressin may be related to obsessive-compulsive disorder symptom severity, while 5-hydroxyindoleacetic acid might be associated with drug response.


Abnormalities in the regulation of vasopressin and corticotropin releasing factor secretion in obsessive-compulsive disorder.

In light of prior data that the central administration of vasopressin in animals is associated with abnormal persistence of behaviors acquired under aversive conditioning, we studied the secretion of arginine vasopressin into the cerebrospinal fluid and plasma in patients with obsessive-compulsive disorder and controls. Patients with obsessive-compulsive disorder had significantly elevated basal levels of arginine vasopressin in the cerebrospinal fluid and significantly increased secretion of arginine vasopressin into the plasma in response to hypertonic saline administration. Moreover, seven of 12 patients with obsessive-compulsive disorder showed a loss of the normal linear relationship between plasma arginine vasopressin level and osmolality. In addition, cerebrospinal fluid corticotropin releasing hormone, which has synergistic effects with arginine vasopressin centrally and at the pituitary gland, was also significantly elevated in patients with obsessive-compulsive disorder compared with controls.


Acute-phase proteins in patients with head and neck cancer treated with interleukin 2/interferon alfa.

Circulating acute-phase proteins may mediate adverse reactions in patients receiving biologic response modifiers, including inhibition of immune responsiveness and clinical toxic effects. Nine patients with unresectable head and neck squamous cell carcinoma were prospectively examined for levels of acute-phase proteins during interleukin 2/interferon alfa immunotherapy and for clinical toxic effects. Simultaneous determination of the in vitro immunomodulatory capacity of autologous serum on the induction of lymphokine-activated killer cells was assessed in 4-hour chromium release assays. Of the seven acute-phase proteins analyzed, haptoglobin and C-reactive protein levels were elevated before therapy was started. Toxic events leading to cessation of interleukin 2/interferon alfa therapy had a high correlation with elevated C-reactive protein and lowered C3 component of complement levels. No relationship was noted between serum levels of acute-phase proteins and induction inhibition of lymphokine-activated killer cell cytotoxicity. The role of C-reactive protein and complement degradation products in mediating interleukin 2/interferon alfa toxicity requires further investigation.


Histochemical and immunohistochemical characteristics of mast cells in nasal polyps.

In surgically excised nasal polyps, most epithelial mast cells were formalin sensitive, chloroacetate esterase (CAE) negative, and chymase negative. Thus, this represents a population of mast cells not identified by staining for CAE. On the other hand, most stromal mast cells were formalin resistant and CAE positive, and although there was some polyp-to-polyp variability in their content of neutral protease, most of these cells were positive for both tryptase and chymase. The percentage of metachromatic cells in the epithelium and the number of metachromatic cells per unit area of polyp tissue did not correlate with an index of allergy skin test reactivity or the serum IgE concentration. The percentage of mast cells surrounded by pericellular tryptase, suggesting activation/degranulation, was significantly higher in the stroma than in the epithelium. The findings demonstrate differences between the stroma and the epithelium in phenotype and state of activation of mast cells; these are postulated to be due to distinct microenvironmental factors that affect mast cells at these sites.


Adhesion molecules on freshly recovered T leukemias promote tumor-directed lympholysis.

Besides facilitating cell to cell adhesion, the molecular interactions between CD2 and its ligand CD58 (lymphocyte function-associated antigen-3 [LFA-3]), as well as between CD11a/18 (LFA-1) and CD54 (intercellular adhesion molecule-1) have recently been recognized to participate in lymphocyte activation, recirculation, and effector function, including cytolytic activity towards tumor cells. We have investigated the role of CD2/CD58 and CD11a/18/CD54 interactions in cellular immune responses directed towards freshly recovered human T-cell leukemias. The data support the notion that downregulation of CD54 and CD58 correlates with enhanced numbers of blasts in circulation and unsusceptibility to killing by autologous cytotoxic lymphocytes. Importantly, after induction of CD54 and CD58 expression on leukemic cells by recombinant cytokines such as tumor necrosis factor-alpha, tumor cells become highly susceptible to lymphocyte-mediated lysis in vitro. Our findings, therefore, stress the point that successful immunotherapy of malignant disease may be facilitated by influencing not only the immune response itself, but also adhesion molecules on the malignant tumor targets.


Factor-dependent erythroid cell lines derived from mice transplanted with hematopoietic cells expressing the v-src oncogene.

Transplantation of spleen cells from primary reconstituted mice expressing the v-src oncogene to secondary and tertiary irradiated recipients resulted in the emergence of erythroid precursors with a transformed phenotype. When cultured in methyl cellulose, these precursors generated colonies of undifferentiated cells that could be expanded into continuously growing factor-dependent cell lines in liquid culture. All lines tested had a similar phenotype and expressed the v-src oncogene. In addition they responded to factors that regulate normal erythroid development, namely erythropoietin (Epo), interleukin-3 (IL-3), and mast cell growth factor (MGF), the ligand to the c-kit encoded receptor. When cells from one of the lines were maintained in the absence of factor, a "factor independent" subpopulation emerged that appeared to grow in an autocrine fashion. Conditioned medium from these cells stimulated their own growth as well as the growth of broad spectrum of normal precursors. Studies with neutralizing antibodies indicated that the predominant colony-stimulating factor produced by these cells is IL-3.


Primary myelodysplastic syndromes: diagnostic and prognostic significance of immunohistochemical assessment of bone marrow biopsies.

Material from 63 cases with primary myelodysplastic syndromes (P-MDS) (French-American-British [FAB] types: refractory anemia [RA] = 21; RA with ring sideroblasts [RARS] = 8; RA with excess of blasts (RAEB) = 10; RAEB in transformation (RAEBt) = 6; chronic myelomonocytic leukemia [CMML] = 10 and unclassifiable = 8, ie, bone marrow aspiration was inadequate and stringent FAB criteria were not applicable) was analyzed for bone marrow histologic and immunohistochemical patterns. Standard Giemsa, hematoxylin and eosin (H&E) and reticulin stains were used for morphologic assessment. To identify the cell lineage precisely, chloroacetate esterase staining and an indirect immunoperoxidase technique using mouse monoclonal antibodies CD15, CD68, HLA-DR, and rabbit polyclonal CD3 and UEA-1 (lectin) was developed on formalin-fixed paraffin embedded bone marrow biopsies (BMB). The immunohistochemical assessment permitted accurate identification of dysplastic features such as mononuclear and binuclear megakaryocytes, Pelger-Huet neutrophils, and binuclear erythroblasts. Additional bone marrow histologic and immunohistochemical features observed were heterogeneity of immunohistochemical staining in various cell lineages, megakaryocytic emperipolesis, alteration of bone marrow microarchitecture, intravascular clusters of hematopoietic cells, and the types of benign lymphoid aggregates. The nature of abnormally localized immature precursors (ALIP) was discerned. Three types of clusters of immature cells were found that were difficult to distinguish on Giemsa and H&E morphology, these were erythroid aggregates (n = 18); megakaryocytic aggregates (n = 4), and immature granulocytic and monocytic aggregates (n = 32). The bone marrow histologic and immunohistologic patterns permitted the identification of four groups of clinical relevance: Group 1, cases with predominant erythroid hyperplasia and without ALIP (n = 15); group 2, cases with prominent myeloid hyperplasia and presence of ALIP (n = 32); group 3, cases with hypoplastic MDS (n = 10); and group 4, cases with hyperfibrotic MDS (n = 6). Statistical analysis showed a significant difference in survival and leukemic transformation between groups 1, 2, 3, and 4, with cases in group 2 showing the worst prognosis with early death due to increased propensity to leukemic transformation and cytopenia-related complications (P less than .0001). We conclude that immunohistochemistry is feasible on routinely processed BMB and the information obtained is of diagnostic and prognostic importance in P-MDS. The phenotype of ALIP varies with the morphologic and histologic subtypes of MDS and the term should be reserved for cases in whom the clusters in the intertrabecular region are of myeloid (granulocytic and monocytic) lineage on immunohistochemistry.


Follicular non-Hodgkin's lymphoma cell adhesion to normal germinal centers and neoplastic follicles involves very late antigen-4 and vascular cell adhesion molecule-1.

Follicular lymphomas recapitulate the architecture of germinal centers (GCs) of normal secondary lymphoid follicles. Using an in vitro binding assay, it has recently been demonstrated that the normal B lymphocytes bind to GCs. This interaction is mediated by a receptor-ligand pair consisting of the beta 1 integrin very late antigen 4 (VLA-4) on the B cell, and the vascular cell adhesion molecule-1 (VCAM-1) expressed on follicular dendritic cells (FDC). Considering the similarities between follicular lymphomas and normal GCs, the adhesive interaction of follicular non-Hodgkin's lymphoma (NHL) cells and GCs was examined. Cells isolated from 16 of 24 cases of follicular NHL bound to normal GCs. Neoplastic follicles could similarly support the binding of follicular NHL cells. This adhesion was inhibited by monoclonal antibodies (MoAbs) directed against VLA-4 and VCAM-1. This supports the hypothesis that the neoplastic follicles used the identical adhesive interactions responsible, at least in part, for the localization of normal B cells to GCs. Adhesion receptors have an important role in the regulation of normal lymphoid cell proliferation, differentiation, and localization. Therefore, an understanding of the adhesive interaction of follicular NHL cells with GCs may provide insight into the clinical and biologic behavior of these diseases.


Absence of abnormalities of c-kit or its ligand in two patients with Diamond-Blackfan anemia.

As Diamond-Blackfan anemia shares clinical features with W and Steel defects in mice, we investigated the possibility that this human disorder might result from an abnormality of the c-kit receptor or its ligand, stem cell factor (SCF). For these studies, full nucleotide sequences for coding regions of c-kit and SCF were generated for two Diamond-Blackfan anemia patients and were normal. Similarly, the kds of SCF receptors on their marrow cells (31 pmol/L, 43 pmol/L) were comparable with those found in three normal controls (50 pmol/L, 55 pmol/L, 27 pmol/L). Serum SCF concentrations were 6.9 ng/mL in patient A, 14.6 ng/mL in patient B, who has been in hematologic remission since adolescence, and 2.7 ng/mL in the 3-year-old daughter of patient B, who also has Diamond-Blackfan anemia but is transfusion-dependent. It is possible that the SCF level in patient B increased with puberty, leading to her remission. These data provide evidence that Diamond-Blackfan anemia does not result from structural abnormalities of c-kit or SCF.


Human lymphokine activated killer (LAK) cells suppress generation of allospecific cytotoxic T cells: implications for use of LAK cells to prevent graft-versus-host disease in allogeneic bone marrow transplantation.

We have found that murine lymphokine activated killer (LAK) cells have veto and natural suppressor activities in vitro, and prevent graft-versus-host disease (GVHD) in vivo. To determine whether human LAK cells mediate veto and natural suppression we measured their ability to inhibit generation of allospecific cytotoxic T cells (CTL) in mixed lymphocyte culture (MLC). When added to MLCs at low concentrations LAK cells caused veto-type inhibition: stimulator-type LAK cells inhibited generation of CTL but responder or third-party LAK cells did not. At higher concentrations LAK cells caused nonspecific inhibition: all three LAK cell types inhibited generation of CTL. LAK cell veto and natural suppressor activities were largely eliminated by irradiation with 30 Gy and by depletion of CD56+ cells, but increased after depletion of CD3+ cells. LAK cell veto activity is not likely an artifact of cold-target inhibition by the LAK cells themselves or by proliferation of T cells contaminating LAK cell preparations: (1) veto only occurred when LAK cells were added to MLC on days 0 through 2, but not when added on day 5; (2) addition of saturating numbers of labeled targets to fixed numbers of allo-CTL effectors failed to overcome the inhibitory effects of adding stimulator-type LAK cells at the onset of MLC; and (3) CD3-depleted LAK cells showed greater veto activity than threefold greater numbers of control LAK cells. In light of our previous findings in mice, the current results imply that adoptive immunotherapy with LAK cells may be useful in preventing GVHD in human bone marrow transplant recipients.


Localization of the platelet-specific HPA-2 (Ko) alloantigens on the N-terminal globular fragment of platelet glycoprotein Ib alpha.

The human platelet-specific alloantigens HPA-2a and HPA-2b (= Kob and Koa) together constitute a biallelic antigen system. The HPA-2 antigens have not, to date, been located on a particular platelet membrane molecule. Here, we describe the localization of these antigens on platelet glycoprotein (GP) Ib alpha. Platelets from two patients with the Bernard-Soulier syndrome (BSS) were HPA-2(a-,b-) in the immunofluorescence test with HPA-2 alloantibodies on chloroquine-treated platelets. With monoclonal antibody (MoAb) immobilization of platelet antigen assay (MAIPA), positive reactions were obtained only when MoAbs against the platelet GPIb/IX complex were used in combination with anti-HPA-2a or -2b alloantibodies and normal donor platelets. By immunoprecipitation under nonreducing and reducing conditions a protein of 160 Kd and 145 Kd, respectively, was precipitated by the anti-HPA-2a serum. A protein migrating identically to this was precipitated by anti-GPIb MoAb. Normal donor platelets became HPA-2(a-,b-) after elastase treatment, suggesting that anti-HPA-2 antibodies bind to the N-terminal elastase-sensitive part of GPIb alpha. Anti-HPA-2a antibodies inhibited the ristocetin-induced agglutination of HPA-2a-positive platelets but not of HPA-2a-negative platelets, indicating that the epitopes recognized by these alloantibodies are localized in the proximity of the von Willebrand-factor-binding domain. Together, these data provide evidence that the HPA-2 alloantigens are located on the N-terminal globular elastase-sensitive part of GPIb alpha. Furthermore, we show that the recently described Siba antigen is probably identical to HPA-2a.


Glucocorticoids downregulate gene expression of GM-CSF, NAP-1/IL-8, and IL-6, but not of M-CSF in human fibroblasts.

Cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage-CSF (M-CSF), neutrophil-activating peptide-1/interleukin-8 (NAP-1/IL-8), and interleukin-6 (IL-6) are pivotal in the regulation of hematopoiesis and immune responses. In mesenchymal cells, their expression is induced by tumor necrosis factor alpha (TNF) and other agents. We now show that, while induction of cytokine expression by TNF in human lung fibroblasts was parallel, glucocorticoid hormones differentially affected their production. Dexamethasone (1 mumol/L) concordantly repressed expression of GM-CSF, NAP-1/IL-8 and IL-6. RNA and protein levels were reduced to approximately 5%, 20%, and 30% of control cells, respectively, as determined by Northern blot analyses and immunoassays. A 50% reduction of RNA levels for all three cytokines occurred in the range of 1 hour. In contrast, dexamethasone (1 mumol/L) did not decrease M-CSF RNA levels and protein release. M-CSF RNA and protein levels were maintained even when dexamethasone (1 mumol/L) was present for the whole duration of a 48-hour TNF stimulation. Further experiments showed that dexamethasone downregulates expression of GM-CSF, NAP-1/IL-8, and IL-6 mainly by decreasing the mRNA stability of these cytokines, and that the dexamethasone-mediated repression of cytokine expression depends on ongoing protein and RNA syntheses. Our study suggests that glucocorticoid hormones repress expression of a set of cytokine genes important in conditions of stress. However, they seem not to affect M-CSF expression, which is likely to be more crucial in maintaining long-term functions of myeloid cells.


Recombinant rat stem cell factor stimulates the amplification and differentiation of fractionated mouse stem cell populations.

The role of recombinant rat stem cell factor (rrSCF) was studied on defined primitive bone marrow cell populations. In agar culture of 500 lineage-negative/Sca-1-positive (Lin-/Sca-1+) cells, rrSCF alone stimulates small colonies of predominantly granulocytic cells. The combinations of rrSCF plus interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or macrophage CSF (CSF-1) stimulated primitive progenitor cells defined as high proliferative potential colony-forming cells (HPP-CFC). Synergistic increases in total colony numbers were obtained with rrSCF plus GM-CSF, granulocyte CSF (G-CSF), CSF-1, or IL-6, but not IL-1 or IL-3. Lin-/Sca-1+ cells were incubated in liquid culture at 3,000 cells/mL for 6 days in the presence of rrSCF alone or in combination with other growth factors. The total number of cells was increased twofold in the presence of rrSCF, with the progeny primarily myeloid in nature. The greatest increase in cell number was obtained with rrSCF plus IL-3, where the cell number increased 40-fold. These factors also stimulated an increase in HPP-CFC (10-fold) and GM-CFC (500-fold). To determine if these interactions were direct, single Lin-/Sca-1+ cells were sorted into microtiter wells and the cell proliferation scored 6 days later. RrSCF synergized with IL-3, IL-6, and G-CSF to stimulate the proliferation of single cells. The cells in positive wells were subcultured into colony-forming assays and up to 400 CFC per well were obtained after 14 days incubation of the secondary cultures. These data demonstrate that rrSCF acts in combination with various growth factors to directly stimulate the amplification potential of hematopoietic primitive precursors, resulting in differentiation of these precursors.


A unique factor XIII inhibitor to a fibrin-binding site on factor XIIIA.

An 81-year-old woman, who presented with sudden episodes of spontaneous bleeding, was found to have a specific inhibitor of factor XIII. Her fibrin clots had approximately 70% gamma-gamma and no alpha polymer formation, under conditions where normal fibrin was fully cross-linked; the patient's clots were soluble in urea or monochloroacetic acid. Factor XIII activity in her plasma was 24%, measured by the dansylcadaverine incorporation assay. When mixed with normal plasma, the patient's plasma inhibited fibrin cross-linking; however, in mixtures of patient and normal plasma, there was no inhibition of factor XIII activity when assayed by the incorporation of dansylcadaverine into casein. Thus, this inhibitor was active against fibrin cross-linking but not against ligation of small molecules to casein. Consequently, gel electrophoresis of reduced, sodium dodecyl sulfate-solubilized fibrin clots was a simple, quantitative method that was used to measure inhibitor activity. This inhibitor is unique and has been designated inhibitor New Haven. It was neutralized by anti-IgG and anti-kappa. It did not inhibit the activation of factor XIII but did inhibit fibrin cross-linking. There was complex formation between the inhibitor and activated factor XIII (A', A*) but not between A2 or fibrinogen. Only A', A* and the 56-Kd fragment bound to affinity columns made with this IgG. The inhibitor significantly decreased the binding of A', A* to fibrin clots. These data indicate that the epitope for this inhibitor is in a fibrin binding site. It is hidden in the zymogen and expressed on A' and A*, indicating that the conformational change occurring with the cleavage of the activation peptide is sufficient to expose the fibrin binding site.


Acute undifferentiated leukemia with CD7+ and CD13+ immunophenotype. Lack of molecular lineage commitment and association with poor prognostic features.

The authors studied six adult patients with acute leukemia with these unusual characteristics: unclassifiable morphology and undifferentiated cytochemistry by French-American-British (FAB) criteria; concurrent expression of CD13 (and CD33) myeloid and early T-cell CD7 immune markers; no evidence of T-cell lineage commitment as determined by T-cell receptor beta (beta), gamma (gamma), and delta (delta) chain gene rearrangement study and cytoplasmic CD3 epsilon expression; and no evidence of myeloid cell lineage commitment, as shown by absent myeloid-specific c-fms proto-oncogene expression and negative myeloperoxidase ultrastructural staining (one case). Clinically, these diagnostic features matched with a poor prognosis, being associated with refractoriness to treatment, relapse and progression of disease, antecedent hematologic abnormality, and other malignancy. These cases may represent a distinct stem cell leukemia syndrome deserving immediate recognition and a nonconventional chemotherapeutic approach.


Bone marrow hypoplasia and aplasia complicating interferon therapy for chronic myelogenous leukemia.

In four patients with Philadelphia chromosome-positive (Ph1) chronic myelogenous leukemia (CML), bone marrow hypoplasia (three patients) and aplasia (one patient) developed during or after therapy with either alpha-interferon (IFN) or gamma-IFN. The predominant clinical characteristic of this complication was protracted pancytopenia, which required 2 to 5 months recovery time after treatment and did not resolve in one patient. Bone marrow cytogenetic analysis in two of the patients demonstrated 100% Ph1 metaphases despite the profound bone marrow suppression. Overall, this complication was uncommon, occurring in less than 2% of the patients with CML treated with various IFN. The possible underlying causes include previous therapy with alkylating agents, lack of "reservoir" or normal stem cells, or pronounced sensitivity of the malignant cell clone to the suppressive effect of IFN.


Establishment of a human t(4;11) leukemia in severe combined immunodeficient mice and successful treatment using anti-CD19 (B43)-pokeweed antiviral protein immunotoxin.

Human acute leukemia, with a chromosomal translocation involving chromosomes 4 and 11, t(4;11)(q21;q23), is the most common form of leukemia in infants and responds very poorly to conventional therapy. A human CD19+ mixed-lineage leukemia cell line with a t(4;11)(q21;q23) translocation, RS4;11, disseminated and proliferated in the hematopoietic tissues and other organs of mice with severe combined immunodeficiency in a manner similar to that observed in humans and killed 100% of the animals. The anti-CD19(B43)-pokeweed antiviral protein immunotoxin selectively inhibited clonogenic RS4;11 cells in vitro, markedly reduced the burden of disseminated leukemia of severe combined immunodeficient mice, and, most importantly, resulted in the long-term survival of treated animals. This severe combined immunodeficient mouse model should be useful for the design of more effective treatment strategies for refractory human leukemias.


The NZB mouse as a model for chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and the only leukemia for which a possible genetic component has been described. Analysis of this genetic component has been hindered by the fact that disease onset normally occurs after age 50. We report here the aged NZB mouse as an animal model for CLL. NZB mice have a genetically regulated, age-dependent onset of clonal, aneuploid cells which are IgM+ and Ly1+ (CD5+ B-cells). Peripheral blood smears from old NZB mice show an increase in circulating lymphocytes and "smudged" or ruptured cells, often seen in human CLL. Electron microscopic examination of these cells shows them to be mature lymphocytes. Light microscopy of the spleen shows infiltration of small lymphocytes and is consistent with CLL pathology. These long-lived, CLL-like cells can be serially passaged into recipient animals. This continued passage occasionally results in the development of a large cell lymphoma detectable in the spleen, lymph nodes, and liver. The histology of this lymphoma is quite distinct from that of the CLL-like cells, but the phenotype is that of an aneuploid CD5+, IgM+ cells. This apparently represents a continued transformation of the CLL-like clone similar to the development of Richter's syndrome in human CLL. Therefore, the NZB mouse can be a valuable tool for the determination of the genetic basis of CLL ontogeny and the conversion of CLL into Richter's syndrome.


Long- and short-term risk of sudden coronary death.

The long- and short-term relation of risk factors to sudden cardiac death (SCD) is examined in the Framingham heart study of 2,011 men and 2,534 women aged 35-70 at the fourth biennial exam. Risk factor measurements over the first four biennial exams were averaged and analyzed as predictors of the long-term occurrence of SCD over the ensuing 28 years using Kaplan-Meier survival curves and the Cox proportional hazards method. The relation of risk factors to the short-term risk of SCD was examined by relating risk factors at each biennium to incidence over the ensuing 2 years, using pooled logistic regression analyses. Over the 28 years of follow-up, 171 men and 80 women experienced SCDs. Women had a lower incidence than men at all ages, and even after adjusting for known risk factors, their SCD rate was only 32% of that in men. In the short term, women have an SCD rate that is 23% of that in men. Most of the modifiable or constitutional risk factors, including glucose intolerance, systolic blood pressure, body mass index, and cigarette smoking have a greater long-term than short-term net effect. This is less apparent in women. Electrocardiographic abnormalities such as left ventricular hypertrophy, intraventricular block, and nonspecific repolarization abnormality were better short-term predictors. In men, preexisting coronary heart disease conferred a 3.3-fold (risk factor-adjusted) increased long-term risk of SCD and 5.3-fold increased short-term risk. In women, the long-term risk is 1.9 and short-term risk is 2.8.(ABSTRACT TRUNCATED AT 250 WORDS)


Dextran administration avoids hemodynamic changes following paracentesis in cirrhotic patients. A safe and inexpensive option.

Paracentesis associated with albumin administration has been shown to be a safe and useful procedure in the treatment of patients with cirrhosis and ascites. Given the high cost of albumin, 20 patients with cirrhosis and ascites were treated in an open study, with daily paracentesis using dextran 70, an inexpensive volume expander, instead of albumin. In the first 10 patients, hemodynamic evaluation was performed in basal conditions, after each paracentesis (5 liter), and after dextran infusion. Twelve hours after each paracentesis without expansion, a significant drop in pulmonary capillary wedge pressure from 9.5 +/- 1.0 to 7.1 +/- 1.7 (P less than 0.01) and a reduction in cardiac output from 6.6 +/- 1.0 to 5.0 +/- 1.9 (NS) were observed. Moreover, the hematocrit rose significantly from 36.8 +/- 5.6 to 39.2 +/- 4.8 (P less than 0.01). These parameters returned to baseline values after the administration of 84 +/- 14 ml of dextran 70 for each 1000 ml of ascites removed. The other 10 patients received dextran 70 simultaneously with the paracentesis without hemodynamic control. No significant changes in renal and hepatic functions were observed at the end of the study. The mean volume of ascites removed was 12.3 +/- 4.6 liter. Two patients developed hyponatremia that required no treatment. No patient developed renal failure. One patient died because of gastrointestinal bleeding.(ABSTRACT TRUNCATED AT 250 WORDS)


A symptomatic female patient with Duchenne muscular dystrophy diagnosed by dystrophin-staining: a case report.

We report a case of symptomatic carrier of Duchenne muscular dystrophy (DMD) in a 14-year-old girl with no prior family history of DMD. She presented with chest pain, enlarged calf muscles, an elevated serum creatine kinase (CK), and decreased left ventricular function. Histological examination of skeletal muscle revealed myopathic changes and immunostaining with anti-dystrophin antiserum demonstrated a mosaic pattern which are compatible with the observations in carriers of DMD. Southern blots using the dystrophin cDNA revealed no evidence of a deletion within the DMD gene in the patient or in her mother. We found this observation interesting for two reasons: 1. Cardiomyopathy is rare in female DMD patients. 2. Immunostaining of a muscle biopsy with anti-dystrophin serum proved to be valuable in the diagnosed for symptomatic carriers of the dystrophin gene mutation.


Regeneration of the eighth cranial nerve in the bullfrog, Rana catesbeiana.

The present study was done in order to document the ability of the eighth cranial nerve of the bullfrog (Rana catesbeiana) to regenerate, the anatomic characteristics of the regenerated fibers, and the specificity of projections from individual endorgan branches of the nerve. The eighth cranial nerve was sharply transected between the ganglion cells and the brain stem in 40 healthy bullfrogs and allowed to regenerate. Anatomic studies were performed in these animals a minimum of 3 months postoperatively. Horseradish peroxidase was used to label the whole vestibular nerve or its individual endorgan branches. Labeled regenerated fibers could be identified crossing the site of the nerve section and projecting centrally to the vestibular nuclei in a pattern similar to that of normal frogs. Labeling of individual branches showed that regenerated fibers innervated the same specific areas found in normal frogs. Unlike normal animals, both thick and thin fibers projected to the medial nucleus.


Does GAP-43 support axon growth by increasing the axonal transport velocity of calmodulin?

GAP-43 is a neuronal protein whose synthesis is elevated during developmental and regenerative axon growth. We propose that one consequence of this increased synthesis may be the delivery of calmodulin-like proteins to the distal portions of the growing axon at an increased velocity; this is because calmodulin, which is transported slowly in mature intact axons, can bind to GAP-43, which is transported rapidly. The release of calmodulin from GAP-43 would be regulated by phosphorylation by protein kinase C. Such a rapid carrier function could be important for allowing certain recently synthesized slowly transported proteins to reach the moving growth cone in time to support its function. This hypothetical carrier mechanism is consistent with the phosphorylation pattern, calmodulin binding, transport velocity, and growth-association of GAP-43, and suggests an explanation for the specific importance of newly synthesized GAP-43 in supporting axon growth.


Fetal cell grafts into resection and contusion/compression injuries of the rat and cat spinal cord.

This article reviews recent findings concerning the feasibility, basic neurobiology, and potential functional benefits of fetal CNS tissue grafts into acute and chronic lesions of the adult spinal cord. In the rat, neuro-anatomical observations suggest that transplants into resection cavities establish neuritic projections that could functionally reunite separated rostral and caudal segments of the host spinal cord. Furthermore, some complementary electrophysiological evidence has been obtained for synaptic connectivity between host and graft neurons. In these studies, extracellular single-unit activity was evoked in fetal spinal cord (FSC) transplants by stimulating host dorsal roots that had been juxtaposed to donor tissue at the time of transplantation. In other investigations, we examined whether grafts could also establish axonal projections to appropriate areas of gray matter in the chronically injured spinal cord. For this purpose, fetal serotoninergic (5-HT) neurons were injected caudal to complete spinal cord transections that had been made 1-3 months earlier. Immunocytochemistry revealed that these cells projected their axons into gray matter regions normally innervated by bulbospinal 5-HT neurons. To investigate transplantation in a more clinically relevant lesion model, a third group of experiments involved injection of dissociated cell suspensions into acute [less than 24 h postinjury (p.i.)]), subchronic (7-10 days p.i), and chronic (greater than or equal to one month, p.i.) contusion lesions. Such grafts routinely filled areas that otherwise would have been regions of cavitation extending rostral-caudal distances of approximately 7 mm. FSC transplants in such injuries also appeared to influence some aspects of motoneuron excitability and hindlimb locomotion. More recent studies of the cat spinal cord have extended these findings in the rat by showing long-term survival (greater than 2 years) of fetal CNS allografts in recipients with either subtotal transection or compression lesions. Preliminary studies of connectivity have also shown host-graft projection patterns similar to those seen in the rat. Behavioral analyses are currently underway to examine the effects of fetal grafts in cats with chronic postcompression lesions. These observations in the rat and cat are discussed in the general context of basic biological and clinical issues relevant to the long-term objective of promoting functional improvement in the damaged spinal cord.


Receptor cell regeneration and connectivity in olfaction and taste.

The capacity of adult mammalian gustatory and olfactory receptor cells to regenerate and make synaptic reconnections provides examples that may be useful in initiating replacement of other kinds of sensory receptor cells. The sensory code for taste quality may not be degraded by taste receptor cell turnover because axons probably recouple to the appropriate type of new receptor cell by axon-receptor cell affinity. Experiments on the development and regeneration of taste receptor cells suggest that they regenerate and turn over by recapitulating the late but not the early steps in taste bud development. To evaluate the replacement of vertebrate olfactory receptors, we began by characterizing the spatial pattern of primary olfactory projections in rainbow trout. Contiguous clusters of HRP-labeled olfactory receptor neurons (ORN) make highly divergent projections to the olfactory bulb. Retrograde transport of fluorescent latex beads revealed that a given restricted site in the glomerular layer received axons from ORNs widely scattered in the epithelium. Hence, ORN axons do not form point-to-point or regional topographic maps. Rather, the olfactory epithelial sheet makes a plane-to-point or holographic-like projection, since any given point in the glomerular layer receives information from the entire olfactory epithelial plane. Receptor cells that reacted with the lectin pokeweed agglutinin were highly dispersed in the olfactory epithelium with axons widely scattered in the olfactory nerve. Yet, as a consequence of the extensive reaggregation of axons at the nerve-bulb interface, the lectin-positive axons fasciculated and converged into a subregion of the glomerular layer.(ABSTRACT TRUNCATED AT 250 WORDS)


Functional properties of acetylcholine receptors coexpressed with the 43K protein in heterologous cell systems.

The nicotinic acetylcholine (ACh) receptor is an integral membrane protein which mediates synaptic transmission at the skeletal neuromuscular junction. A key event in the development of the neuromuscular junction is the formation of high density aggregates of ACh receptors in the postsynaptic membrane. Receptor clustering has been attributed, in part, to their association with a peripheral membrane protein of Mr 43,000 (43K protein). We have addressed whether the association of the 43K protein can alter the single channel properties of the ACh receptor, and thus influence neuromuscular transmission at developing synapses, by expressing ACh receptors with and without the 43K protein in heterologous expression systems. We found that coexpression of the 43K protein with the receptor did not significantly alter either its single channel conductance or its mean channel open time. This was true in oocytes and also in COS cells where it was possible to localize 43K-induced clusters by fluorescence microscopy and to record from those clustered receptors. These data are in agreement with previous single channel studies which have shown that the properties of diffusely distributed and clustered receptors in native muscle cells from both mice and Xenopus do not differ.


Avian scale development. XVI. Epidermal commitment to terminal differentiation is prior to definitive scale ridge formation.

Germinative cells of the scutate scale epidermis from 15-day embryos are committed to appendage-specific, beta stratum formation in association with a foreign dermis. Commitment precedes the time (17 days of development) at which beta strata are actually present in their site-specific locations along the outer surface of each scutate scale. This observation suggested the possibility that commitment to beta stratum formation might be occurring as the outer epidermal surface of each scutate scale first becomes established between 12 and 13 days of development. It is at this time that the scale epidermis loses its ability to participate in feather morphogenesis and cell proliferation becomes restricted to a true stratum basale. To examined the ability of the presumptive scutate scale epidermis to generate beta strata in the absence of the inductive scutate scale dermis, scutate scale epidermis from 11-, 12-, and 13-day embryos was recombined with 15-day reticulate scale dermis and grown for 7 or 9 days. The dermis of reticulate scales does not induce beta stratum formation, but it does support differentiation of a beta stratum by the determined 15-day scutate scale epidermis. Using immunohistological and biochemical analyses of beta-keratins, we find that each of these presumptive scutate scale epidermises is competent to generate appendage-specific beta strata in the absence of the scutate scale dermis. This determination is occurring prior to scale ridge morphogenesis and differentiation of the epidermis into the distinct outer and inner epidermal surfaces of the scale ridge. The restricted distribution of beta strata to the apical domes of individual reticulate-like scales demonstrates that the germinative cells of the committed epidermises are responding to patterned cues. This study also suggests that all basal cells of the presumptive scutate scale epidermis are initially endowed with the ability to generate cells that form a beta stratum.


Immunization against polyoma tumors with synthetic peptides derived from the sequences of middle- and large-T antigens.

We have used 9 synthetic peptides corresponding to sequences of polyoma virus small-T, middle-T and large-T antigens as immunogens in order to map antigenic epitopes that can induce polyoma-tumor-specific immunity in different mouse strains. We found that immunization of mice with synthetic peptides derived from amino acid (aa) sequences common to all 3 T-antigens (aa 1-19), or sequences common to only middle-T and small-T (aa 162-176), as well as synthetic peptides unique for middle-T (aa 269-282 and 371-381), or unique for large-T (aa 108-124, 316-333 and 436-449) can induce immunity against polyoma tumors. The synthetic peptides can be divided into 3 types with regard to immunogenicity; (i) peptides that immunize in more than one mouse strain and may represent immunodominant sites, (ii) peptides that may be immunogenic in only one strain, and thus strain-specific, and finally (iii) peptides that do not immunize in the strains tested so far.


Haemopoietic cell kinetics in humans treated with rGM-CSF.

We have investigated the kinetics of myeloid cell proliferation in the marrow of patients with small-cell lung cancer and treated with 10 daily subcutaneous injections of granulocyte/macrophage colony-stimulating factor (GM-CSF). Bone marrow, obtained before and during treatment with the growth factor, was labelled in vitro with tritiated thymidine (3H-TdR). A 3rd bone-marrow sample was obtained 1 hr following an intravenous injection of 3H-TdR. Subsequent daily blood samples were also collected, and 3H-TdR labelling was assessed on these and the marrow preparations by autoradiography. GM-CSF treatment increased the peripheral granulocytic cells nearly 5-fold, but this included significant eosinophilia, so that the neutrophilic granulocytes increased only 3.3-fold. These cells were released from the marrow over a normal time scale, but their peripheral half-life was about 6 times longer than normal and they were probably functionally defective. Furthermore, significant numbers of immature cells were released from the marrow. Neutrophil production stimulated by GM-CSF was thus overestimated by measurement of the apparent peripheral granulocytosis. Increased labelling indices and grain counts in the proliferating granulocytic cells of the marrow indicate shortened cell-cycle times, and the excess granulocyte production appears to be the result of extra amplification divisions in the proliferative compartments.


Effects of interferons on the expression of the proto-oncogene HER-2 in human ovarian carcinoma cells.

The over-expression of the proto-oncogene HER-2 (c-erbB-2/neu) in ovarian, endometrial and mammary carcinoma is an important indicator for poor prognosis. We have previously shown in 3 out of 4 ovarian carcinoma cell lines an interferon-gamma (IFN-gamma)-mediated reduction in HER-2 specific protein and RNA levels. The oncogene expression was lowered only in the ovarian carcinoma cell lines but not in 3 IFN-gamma-sensitive human breast cancer cell lines. We extended our observations also to IFN type I, alpha and omega. The expression of the oncogene was measured by both the p185HER-2 ELISA and in selected cases by a living cell radioimmunoassay using the monoclonal antibody (MAb) 4D5 against the extracellular domain. Both IFN types reduced the expression of HER-2 in the ovarian carcinoma cell lines OVCAR-3, HTB-77, 2774 and SKOV-6, and in the SKUT-2 endometrial carcinoma cells. In contrast, SKOV-8 human ovarian carcinoma cells were sensitive for both IFN types regarding proliferation, but only IFN-gamma reduced proto-oncogene expression. In the SKBR-3 human mammary carcinoma cells, neither IFN type had an effect on HER-2 expression. The antibodies 4D5, 7C2, 3E8, and 3H4 which bind to the extracellular domain of p185HER-2 protein specifically inhibited anchorage-independent growth of SKBR-3 and HTB-77 cells. Expression of the oncogene HER-2 is the leading prognostic factor in ovarian cancer. Its modulation might represent a mechanism by which IFNs inhibit cell proliferation.


Localization of epidermal growth factor immunoreactivity in sheep skin during wool follicle development.

Interactions among the cells and matrices of the epidermis and mesenchyme of skin are essential for hair follicle initiation and development. The identification of receptors for epidermal growth factor (EGF) on epithelial components of the follicle during growth has suggested that the ligand participates in some of these events. We have used affinity-purified antibodies together with an alkaline phosphatase detection procedure to investigate the distribution of EGF in the skin of the sheep during wool follicle formation. Immunoreactivity was restricted to the periderm and intermediate layers of fetal epidermis at 55 d of gestation, when the first wave of wool follicles are initiated. This particular distribution persisted during subsequent development but never became associated with the basal cells of the epidermis. The activity was lost around 118 d, coinciding with sloughing of the periderm. No immunoreactivity was found in the plugs or the dermal condensations of the developing follicles. At approximately 105 d of gestation, however, reactions were detected in the outer root sheath as the follicles matured and in the differentiating cells of the sebaceous glands. A similar distribution pattern was also noted at 140 d, just prior to birth, and in adult animals, indicating that EGF was sequestered and perhaps synthesized within the follicle. The presence of immunoreactive material was also associated with the pilary canals and the skin surface, suggesting that this may have had its origin in the sebaceous glands. We examined this using a radioreceptor assay for EGF. Material washed from the skin surface and sebaceous gland extracts were found to displace 125I-EGF from rat liver membranes, in parallel with mouse EGF.


Central rod domain insertion and carboxy-terminal fusion mutants of human cytokeratin K19 are incorporated into endogenous keratin filaments.

Keratins comprise a multigene family of structural proteins that form the 10-nm filaments present in epithelial cells. Keratin filament formation requires the presence of stoichiometric quantities of type I and type II keratin peptides. Each keratin peptide contains an N-terminal "head" segment, a C-terminal "tail" segment, and a highly conserved, alpha-helical central rod domain. To investigate the importance of these domains in situ, we have altered the DNA coding sequence of human cytokeratin K19 and transiently expressed the mutants in PtK2 cells that contain an endogenous keratin filament system. Interestingly, K19 mutants containing 4, 8, 12, and 24 amino acid insertions in the non-alpha-helical L1 region of the central rod domain successfully integrate into the endogenous PtK2 keratin filaments. Another K19 mutant, K19-bGAL, that encodes bacterial beta-galactosidase (bGAL) fused in phase to the 3' end of the K19 central rod domain, also integrates into the endogenous PtK2 keratin filaments. Our results demonstrate 1) that the spacing between the highly conserved amino and carboxy terminal ends of the K19 central rod domain can be increased without significantly effecting K19's ability to interact with keratin filaments and 2) that addition of a highly soluble 66-kDa tail to K19 does not impede its interaction with the filament system.


Culture of basal cell carcinoma.

Studies of basal cell carcinoma have been hindered by a lack of a suitable and reproducible tissue-culture model system. We have succeeded in growing this tumor in primary culture from eight different patients. We can separate and grow the tumor cells and the stromal cell component. We also culture normal keratinocytes and fibroblasts from the same patient for comparative studies. All the cell types have been subcultured four to five times and cryopreserved. The normal keratinocytes were indistinguishable from the tumor cells in ploidy, in rate of growth, and in the failure to express ICAM-1. Both cell types also fail to synthesize the matrix proteins: types I and IV collagens. Differences were noted in the expression of fibronectin and the bullous pemphigoid antigen, with the normal cells expressing the antigens although the tumor cells did not. Interferons exogenously added to the culture media preferentially killed the basal cell carcinoma cells, as compared to normal keratinocytes from the same patients. We believe that our culture system opens possibilities for biochemical and molecular studies of this disease, and for in vitro testing of antitumor agents for clinical therapy.


Expression of the L-fucose moiety on infrainfundibular follicular keratinocytes of terminal follicles, its decreased expression on vellus and indeterminate follicles of androgenetic alopecia, and re-expression in drug-induced hair regrowth.

The distribution of various glycoprotein molecules on the surface of follicular keratinocytes was studied with a panel of lectins with specificity for various sugar moieties on biopsy specimens from both bald/balding scalp and normal occipital scalp, of 23 patients with androgenetic alopecia as well as on biopsies of normal forearm skin of four patients. The most significant differences between bald and normal scalp biopsy were noted with Ulex europaeus agglutinin I (UEA I). We noted an increased (91.8% +/- 3.1; mean +/- SE) expression of UEA I binding sites on the infra-infundibular follicular keratinocytes in anagen terminal scalp hairs, compared to 28.5% +/- 5.2 in the indeterminate (anagen) hairs of balding scalps, and 23.2% +/- 6.3 in the anagen follicles of vellus fore-arm hairs. By contrast, the telogen hairs demonstrated minimal UEA I staining: 4.0% +/- 0.8, mean +/- SE in telogen scalp hairs, 1.8% +/- 0.5 in telogen hairs of balding scalps (0% in completely bald scalps, in which all the hairs were in the telogen phase), and 1.9% +/- 0.2 in telogen forearm hairs. The percentage of UEA I staining correlated with the length of the infra-infundibular follicles in all cases studied. In three cases of hair regrowth after hair growth promotors, the UEA I staining increased to 80.6% +/- 6.1 in anagen hairs and correlated with increased length of infra-infundibular follicles. Our data indicate that there are 1) marked differences between anagen and telogen follicles in UEA I binding to infra-infundibular follicular keratinocytes; 2) the percentage of UEA I staining reflects the size (length) of the infra-infundibular hair follicle; and 3) the anagen follicles of balding scalps (indeterminate hairs) show UEA I staining resembling that exhibited by anagen follicles of vellus hairs.


VCAM-1-, ELAM-1-, and ICAM-1-independent adhesion of melanoma cells to cultured human dermal microvascular endothelial cells.

We have examined the mechanisms by which tumor cells bind to endothelial cells utilizing cultured melanoma cells and microvascular endothelial cells derived from human dermis (HDMEC). The ability of biologic response modifiers (BRM) to modulate the adhesion of melanoma cells to HDMEC was defined and those results were compared with results from human umbilical vein endothelial cells (HUVEC). SK-MEL-2, WM266-4, and Hs 294T melanoma cells all bound to HDMEC and HUVEC monolayers and adherence of melanoma cells was enhanced in a dose- and time-dependent manner by the treatment of HDMEC with interleukin 1 (IL-1) alpha or tumor necrosis factor (TNF) alpha. Similar increases in binding to HDMEC or HUVEC were induced after BRM stimulation, although baseline melanoma cell binding to HUVEC tended to be slightly higher than to HDMEC. In contrast, whereas phorbol 12-myristate 13-acetate (PMA) augmented melanoma cell adherence to HDMEC, PMA failed to increase adherence to HUVEC. The alterations in melanoma cell binding were induced only after pretreatment of endothelial and not melanoma cells with PMA. Studies of the expression of cell adhesion molecules (CAM) on HDMEC and HUVEC using enzyme-linked immunosorbent assay showed that vascular cell adhesion molecule 1 (VCAM-1) is not induced by PMA on HDMEC and intercellular adhesion molecule 1 (ICAM-1) is downregulated on HDMEC by PMA treatment. Endothelial leukocyte adhesion molecule 1 (ELAM-1) is induced by PMA, IL-1 alpha, or TNFalpha, but its expression does not correlate with increased melanoma cell binding MoAb recognizing VCAM-1-inhibited TNFalpha-induced increases in melanoma cell binding to HUVEC. However, anti-VCAM-1 antibody failed to clock melanoma cell binding to PMA or IL-1 alpha-stimulated HDMEC and only partially inhibited melanoma cell binding to TNF alpha-stimulated HDMEC. This study demonstrates that PMA and IL-1 alpha-induced increases in melanoma cell adherence to HDMEC are not mediated via known CAM, including ICAM-1, VCAM-1, or ELAM-1, and may be affected through microvessel-specific novel proteins not previously described on endothelial cells.


Comparison of the University of Wisconsin preservation solution and other crystalloid perfusates in a 30-hour rabbit lung preservation model.

The University of Wisconsin solution, which contains a high potassium concentration (120 mmol/L), was evaluated for rabbit lung preservation by comparing it with a modified University of Wisconsin solution with low potassium (4 mmol/L), a low-potassium dextran solution (4 mmol/L), and simple surface cooling. In the first three groups rabbit lungs were flushed in situ with the solution (n = 5 in each group); then the lung-heart block was harvested and stored at 10 degrees C for 30 hours. In the surface cooling group the lungs were harvested without flushing and then simply immersed in saline and stored. For assessment, the stored lung was ventilated with room air and perfused with fresh venous blood at a rate of 40 ml/min for 10 minutes. Assessment of lung function included gas analysis of effluent blood, mean pulmonary artery perfusion pressure, and peak airway pressure. Among these parameters, oxygen tension was most sensitive. Oxygen tension at 10 minutes' perfusion in the modified University of Wisconsin (95 +/- 6 mm Hg) and low-potassium dextran (99 +/- 4 mm Hg) groups was significantly higher than that in the surface cooling (61 +/- 7 mm Hg) and University of Wisconsin (51 +/- 7 mm Hg) groups. There was no difference between the modified University of Wisconsin and low-potassium dextran groups or between the surface cooling and University of Wisconsin groups. We conclude that the low-potassium University of Wisconsin solution is superior to the high-potassium University of Wisconsin solution and that the lactobionate and raffinose included in the University of Wisconsin solution as impermeants do not improve lung preservation in this model.


Cutaneous responses to vasoactive intestinal polypeptide in chronic idiopathic urticaria.

Cutaneous wheal and flare responses to increasing concentrations of calcitonin gene-related peptide, substance P, neurokinin A, vasoactive intestinal polypeptide (VIP), compound 48/80, and phosphate-buffered saline were measured in 10 patients with chronic idiopathic urticaria and 10 healthy controls. A significant increase in VIP-induced wheal, but not flare or cutaneous blood flow, was seen in urticarial patients compared with controls (p less than 0.001). No significant differences in responses to other tested compounds were found between these groups. These data point to an increased sensitivity of microvasculature to VIP in patients with chronic idiopathic urticaria.


Membrane dihydrolipoamide acetyltransferase (E2) on human biliary epithelial cells in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is associated with serum antibodies that react with the dihydrolipoamide acetyltransferase component (E2) of the mitochondrial pyruvate dehydrogenase complex. We have sought the presence of E2 on the surface of human intrahepatic biliary epithelial cells (BEC). Cultured BECs from PBC but not normal liver were found to have E2 on the membrane after three days' culture. Isolated, viable cells examined by laser-scanning confocal microscopy revealed the pattern of E2 staining on the membrane to be similar to that seen with the membrane glycoprotein marker, HEA-125. By contrast, BECs from normal liver showed membrane staining only with HEA-125. When BECs were fixed before incubation with antibody to E2, cytoplasmic staining was observed. Our results suggest that E2 is present on the surface of biliary epithelial cells in PBC, and support the idea of a pathogenetic association between antimitochondrial antibodies and bileduct damage.


Cytosolic activation of 2-aminoanthracene: implications in its use as diagnostic mutagen in the Ames test.

The metabolic activation of 2-aminoanthracene to mutagens in the Ames test was investigated using hepatic S9, microsomal and cytosolic fractions from control and Aroclor 1254-treated rats as activation systems. Microsomal and S9 preparations from control animals could activate 2-aminoanthracene, but the efficiency of activation was suppressed by pretreatment of animals with Aroclor 1254. Cytosolic fractions from Aroclor 1254-treated rats could readily activate the promutagen more readily than microsomes. The cytosolic activation of 2-aminoanthracene required NADPH and could not be accounted for by possible microsomal contamination. The molybdenum oxygenases appear not to contribute to the cytosolic activation of this promutagen. It is concluded that (a) the microsomal activation of 2-aminoanthracene is catalysed more effectively by enzyme systems other than the P450 I family and (b) an enzyme system capable of activating this carcinogen in vitro is present in the hepatic cytosol. The implications of these findings in the use of 2-aminoanthracene as a positive control in the Ames test are discussed.


Induction of the cytoplasmic 'petite' mutation by chemical and physical agents in Saccharomyces cerevisiae.

A range of physical and chemical agents induce the mitochondrial 'petite' mutation in the yeast Saccharomyces cerevisiae. DNA intercalating agents as well as chemicals which can interfere with DNA synthesis induce this mutation, but only in growing cells. Many chemical or physical agents that produce a DNA lesion which is not simply reversed can induce various levels of the petite mutation, and may be more effective in non-growing cells. A limited number of chemicals act like ethidium bromide, inducing a high frequency of petites which is partially reversible with increasing concentration or time. The ability of a specific compound to be transported into mitochondria or its affinity for AT base pairs in DNA may determine whether it acts primarily as a nuclear or mitochondrial mutagen. In mammalian cells, some neoplastic changes occur at the mitochondrial level. Analogies between yeast and mammalian mitochondria suggest that agents which increase petite mutagenesis in yeast may have some carcinogenic potential. Although some types of petite inducer may have potential as antitumour drugs, those which are very effective antimitochondrial agents appear to be too toxic for therapeutic use. A process comparable to early stages in petite mutagensis occurs in human degenerative diseases and it seems possible that a consequence of exposure to petite mutagens could be an increase in the rate of degenerative diseases or of the aging process.


Stable expression of monkey cytochrome P-450IA1 cDNA in Chinese hamster CHL cells and its application for detection of mutagenicity of aflatoxin B1.

A monkey cytochrome P-450IA1 cDNA (MKah1) was transfected into Chinese hamster CHL cells using a vector containing the SR alpha promoter, and sublines stably expressing P-450IA1 were established. The cells showed 25-fold higher sensitivity to the cytotoxic effect of aflatoxin B1 than the parental CHL cells. This hypersensitivity was almost completely suppressed by alpha-naphthoflavone, which is a known specific inhibitor of P-450IA. The cells expressing P-450IA1, but not CHL cells, showed a positive response to aflatoxin B1 in an assay for mutagenicity at the HGPRT locus.


Effect of sampling time on chromosome aberration yield for 7 chemicals in Chinese hamster ovary cells.

Choice of harvest time is one of the most important variables in the assessment of whether a compound is clastogenic and in establishing a dose relation. We examined the effects of sampling time on aberration yield for 7 diverse chemicals in CHO-WBL cells by harvesting at intervals from 9 to 30 h after treatment for 3 h with or without S9 metabolic activation. We observed both the percentage of aberrant cells and the total number of aberrations. Our data suggest that for most compounds a single harvest time approximately 17-21 h after the beginning of a 3-h treatment is optimal for aberration detection in CHO cells. Maximal aberration yields were observed for 2,4-diaminotoluene, 2,6-diaminotoluene and cytosine beta-D-arabinofuranoside from 17 to 21 h, eugenol from 15 to 21 h, cadmium sulfate from 15 to 24 h and 2-aminobiphenyl, from 17 to 24 h. For adriamycin at 1 microM, the % aberrant cells remained elevated throughout the period from 9 to 29 h, while small increases at 0.1 microM ADR were found only at 13 and at 25 h. For most chemicals the maximal aberration yield occurred at a different time for each concentration tested. However, the use of 3 or more closely spaced concentrations, carefully selected to yield up to 50% toxicity, allowed detection of a positive response at a single harvest time for all 7 chemicals.


A quantitative assessment of the cytotoxicity associated with chromosomal aberration detection in Chinese hamster ovary cells.

Regulatory guidelines suggest testing chemicals up to cytotoxic doses in chromosomal-aberration assays. To investigate the utility and limitations of various cytotoxicity indicators we used Chinese hamster ovary (CHO) cells to test 8 chemicals with differing ratios of cytotoxicity to clastogenicity. We measured immediate or delayed cell killing and growth inhibition (ATP levels, cell counts, colony-forming efficiency, CFE) and cell-cycle perturbations (mitotic index, MI; average generation time, AGT). Aberrations (abs) were scored 10 and 24 h from the beginning of the 3-h treatment. All 8 compounds induced abs at concentrations that reduced cell growth at 24 h by 50% or less. Concentrations of each chemical which induced at least 15% cells with abs, gave little loss of CFE (0-20%) for mitomycin C, adriamycin, cadmium sulfate and 2,6-diaminotoluene in contrast to the marked loss of CFE (70-80%) for eugenol (EUG), 2-aminobiphenyl and 8-hydroxyquinoline (8-HQ). 2,4-Diaminotoluene (2,4-DAT) was intermediate. Higher aberration yields were found at 24 h than at 10 h, even when minimal cell-cycle delay was detected by AGT estimates from BrdUrd-labeled cells. Cells with multiple abs were seen at 24 but not at 10 h, and often confirmed clastogenicity when there was only a weak increase in the percentage of cells with aberrations. Total ATP per culture did not always correlate with cell number, especially at later times after treatment. This is likely due to metabolic perturbations or altered cell biomass that are known to affect cell ATP content. MI suppression often did not correlate with AGT, e.g., only small increases in AGT were seen for 8-HQ, 2,4-DAT and EUG despite severe mitotic suppression at 10 h. By 24 h the MI for all chemicals had recovered, sometimes exceeding control levels. Marked mitotic accumulation was seen at 10 h for 2,4-DAT, indicating cell synchrony. Thus, the MI has limited value for dose selection. In conclusion, even weakly active chemicals were detected at a single time without exceeding a 50% growth reduction at 24 h.


Structural requirements for the induction of the SOS repair in bacteria by nitrated polycyclic aromatic hydrocarbons and related chemicals.

The CASE (computer-automated structure evaluation) methodology was used to investigate the structural basis of the SOS-inducing activity of 56 nitrated polycyclic aromatic hydrocarbons (nitroarenes, nPAH) and the unsubstituted parent PAH molecules. Based upon the presence and/or absence of structural features, CASE identified 5 activating (biophores) and 4 inactivating (biophobes) fragments responsible for the SOS-inducing activity. Based upon these fragments, CASE correctly calculated the genotoxicity of 94.6% of the molecules in the training set (sensitivity = 0.85, specificity = 1.0). Disregarding the questionable experimental results of the unexpected very weak direct-acting activity of the unsubstituted benzo[a]pyrene, dibenzo[a,h]anthracene and 7,12-dimethylbenz[a]anthracene, the concordance of the prediction was 100%, i.e., sensitivity = 1.0, specificity = 1.0. Additionally, the quantitative analysis of the SOS-inducing potency showed a good correlation between the experimental and predicted results. The present analyses indicate an identity in the structural determinants responsible for SOS induction in E. coli PQ37 (SOS chromotest) and mutagenicity in Salmonella typhimurium.


Oxygen species and the genotoxicity of quercetin.

Quercetin has been extensively studied in various short-term assays for genotoxicity. The patterns of genotoxicity of quercetin for different genetic endpoints are subject to a variety of factors (pH, antioxidants, metabolism) whose precise role in each test remains unclear. In the present study we report on the possible effect of oxygen-derived species on the activity of quercetin in the Ames assay and in the SOS chromotest. Our results seem to suggest that superoxide dismutase (SOD) does not account for the levels of mutagenicity detected in the presence of S9 or S100. The latter may, however, contain other factors of antioxidant defense which may prevent the oxidative degradation of quercetin. Since this degradation occurs at pH values above neutrality and the SOS-inducing activity is higher at pH 6.0, it is concluded that the response of quercetin in the SOS chromotest is due to quercetin itself at acidic pH. The SOS-inducing activity at pH 7.4 is enhanced by SOD, but it cannot be unambiguously concluded that this effect in the SOS chromotest might only be due to protection against the oxidative degradation of quercetin.


Involvement of RecB-mediated (but not RecF-mediated) repair of DNA double-strand breaks in the gamma-radiation production of long deletions in Escherichia coli.

Experiments were designed to determine the association between the repair of gamma-radiation-induced DNA double-strand breaks (DSB) and the induction of 700-1000 bp long deletions (Lac(-)----Lac+), base substitutions (leuB19----Leu+), and frameshifts (trpE9777----Trp+) in Escherichia coli K-12. Over the range of 2.5-20 krad, deletions were induced with linear kinetics, as has been shown for the induction of DSB, while the induction kinetics of base substitutions and frameshifts were curvilinear. Like the repair of DSB, deletion induction showed an absolute requirement for an intact recB gene as well as a dependency on the type of preirradiation growth medium; these requirements were not seen for base substitutions or frameshifts. In addition, about 80% of the spontaneous deletions were absent in the recB21 strain. A recC1001 mutation, which confers a 'hyper-Rec' phenotype, increased the rate of gamma-radiation-induced deletions as well as the low-dose production of base substitutions and frameshifts. A recF143 mutation increased the yield of gamma-radiation-induced deletions without increasing base substitutions or frameshifts. A mutS mutation markedly enhanced the gamma-radiation induction of frameshifts, and had a slight effect on base substitutions, but did not affect the induction of deletions. Resistance to gamma-irradiation and the capacity to repair DSB (albeit at about half the normal rate) were restored to the radiosensitive recB21 strain by the addition of the sbcB21 and sbcC201 mutations. However, the radioresistant recB sbcBC strain, which is recombination proficient via the RecF pathway, was still grossly deficient in the ability to produce deletions. A model for deletion induction as a by-product of the recB-dependent (Chi-dependent) repair of gamma-radiation-induced DSB is discussed, as is the inability to detect deletions in cells that use only the recF-dependent (Chi-independent) mechanism to repair DSB.


Chromosome rearrangements associated with CAD gene amplification. Experiments with cell hybrids.

Resistance to phosphonacetyl-L-aspartate (PALA) is caused by CAD gene amplification. The marker chromosome of a PALA-resistant cell line containing a homogeneously staining region with amplified CAD gene was introduced into PALA-sensitive Chinese hamster cells by microcell-mediated chromosome transfer. Two monochromosomal hybrids containing the marker chromosome in addition to the normal chromosome complement of sensitive cells and 1 tetraploid hybrid containing the complete genomes of donor (resistant) and recipient (sensitive) cells were studied in detail. It was shown that (i) the presence of the marker chromosome was both a necessary and a sufficient condition for the expression of the PALA-resistant phenotype; (ii) the marker chromosome underwent rearrangements in the monochromosomal hybrids, with preferential loss of non-amplified chromosomal regions, while it was not rearranged in the tetraploid hybrid; (iii) unlike the original PALA-resistant cells obtained after long-term selection in the presence of PALA, the PALA-resistant hybrids did not show chromosomal aberrations of other than the marker chromosome. This result indicates that chromosomal aberrations may be due to the selective procedure and is not an inherent property of cells containing amplified genes.


Allergic rhinitis. Measures to control the misery.

Chronic fatigue, recurrent otitis and sinusitis, and poor performance at school or at work are among the many physical and psychosocial consequences of allergic rhinitis. Antihistamines, decongestants, and nasal sprays may be used to treat many patients, but immunotherapy may be necessary or more practical in others. In all cases, control of the patient's home environment, particularly the bedroom, is essential.


Value of balloon dilation in treatment of youthful patients with prostatism.

Forty-three youthful patients with uncomplicated prostatism were prospectively evaluated to test the safety and efficacy of transurethral balloon dilation (TUDP). Treatment consisted of cystoscopic placement of an intraprostatic balloon inflated to 25 mm diameter at 3 atm pressure for ten minutes. At longest follow-up (9.8 months, average; 3-24 months, range), 88 percent of patients were satisfied with overall treatment results. The average improvements in voiding symptom score and peak uroflow were 77 percent and 73 percent, respectively. Mean improvements over pretreatment levels were statistically significant at one month (p less than 0.01) and remained so for the entire follow-up period. No incontinence, impotency, retrograde ejaculation, sepsis, or serious bleeding developed. An intraprostatic fissure, which spared the bladder neck, was a uniform finding and the most likely mechanism of lasting action of TUDP. In the relief of uncomplicated prostatism in youthful patients, TUDP compares favorably with other treatment alternatives.


A new brain glucosensor and its physiological significance.

The concentration of fibroblast growth factor (FGF), which is found in cerebrospinal fluid (CSF), markedly increases after the start of feeding. Food intake was dose-dependently suppressed by picomole doses of FGF and facilitated by anti-FGF antibody. This suppression was caused by activation of protein kinase C in glucose-sensitive neurons in the lateral hypothalamus. In situ hybridization by use of cDNA showed that acidic (a)FGF was produced in ependymal cells. The ependymal cells released aFGF by responding to glucose increase in CSF after feeding. Released aFGF diffused into the brain parenchyma and was taken by neurons. Passive avoidance was significantly more reliable after aFGF infusion into CSF. Clamping cerebral arteries in the gerbil induced ischemia, which damaged neurons in the CA1 layer of the hippocampus. Pretreatment with aFGF prevented this damage. Thus, aFGF is not only the most potent substance yet found for the suppression of feeding, but it is also extremely effective as a neurotrophic and memory facilitating substance.


Insulin signal transduction: the role of protein phosphorylation.

Recent evidence suggests that the mechanism of insulin action depends in part on protein phosphorylation on tyrosine residues. A cascade of phosphorylation/dephosphorylation reactions is proposed to modulate multiple enzymes involved in metabolism, protein synthesis, and cell growth. Direct evidence is presented for the phosphorylation of myelin basic protein and microtubule-associated protein 2 on tyrosine residues by the insulin receptor.


A new generation of animal cell expression vectors based on the Semliki Forest virus replicon.

We have developed a novel DNA expression system, based on the Semliki Forest virus (SFV) replicon, which combines a wide choice of animal cell hosts, high efficiency and ease of use. DNA of interest is cloned into SFV plasmid vectors that serve as templates for in vitro synthesis of recombinant RNA. The RNA is transfected with virtually 100% efficiency into animal tissue culture cells by means of electroporation. Within the cell, the recombinant RNA drives its own replication and capping and leads to massive production of the heterologous protein while competing out the host protein synthesis. The expression system also includes an in vivo packaging procedure whereby recombinant RNA is packaged into infectious virus particles using cotransfection with packaging-deficient helper RNA molecules. The resulting high titer recombinant virus stock can be used to infect a wide range of animal cells with subsequent high expression of the heterologous gene product, but without expression of any structural proteins of the helper. The infected cells produce protein for up to 75 hours post infection after which the heterologous product can constitute as much as 25% of the total cell protein. The general utility of the system is demonstrated through the expression of human transferrin receptor, mouse dihydrofolate reductase, chick lysozyme and Escherichia coli beta-galactosidase.


Stimulator cell-dependent requirement for CD2- and LFA-1-mediated adhesions in T lymphocyte activation by superantigenic toxins.

The staphylococcal enterotoxins and related microbial T cell mitogens stimulate T cells by cross-linking variable parts of the T cell receptor (TCR) with MHC class II molecules on accessory or target cells. We have used cloned human T cells and defined tumor cells as accessory cells (AC) to study the requirements for T cell activation by these toxins. On AC expressing high levels of CD54 (intercellular adhesion molecule-1, ICAM-1) and CD58 (lymphocyte function-associated antigen-3, LFA-3), mAb to CD2 were relatively ineffective in inhibiting the response to the toxins and antibodies to the lymphocyte function-associated antigen-1 (LFA-1) did not inhibit at all. If added together, however, these mAb inhibited the response completely. Similar results were obtained using antibodies to the target structures of CD2 and LFA-1. In contrast, on cells expressing low levels of LFA-3, mAb to LFA-1 but not to CD2 were strongly inhibitory. The same pattern of inhibition was found when these same cells were used as presenters of specific antigen to the T cells. These data show that adhesions via CD2 or LFA-1 are alternatively required for the stimulation of the T cells by superantigenic toxins and demonstrate another similarity between T cell stimulation by superantigens and by specific antigen recognition.


Regulation of myelin basic protein-specific helper T cells in multiple sclerosis: generation of suppressor T cell lines.

Suppressor T cell (Ts) lines specific for myelin basic protein (MBP)-reactive helper T cell (Th) clones were generated from two patients with multiple sclerosis (MS) following a primary culture of peripheral blood mononuclear cells (PBMC) with MBP and cyclosporine A (CsA). These suppressor T cell lines were maintained in culture by alternate stimulation with MBP and antigen-presenting cells (APC). The Ts lines expressed preferentially the CD4 phenotype (5/6 Ts lines tested) and exhibited potent antigen-specific suppressor activity on the proliferation of MBP-specific Th clones and not on the T cell lines with other antigen specificity. For some Ts lines, a Ts-to-Th ratio of 1 was sufficient to inhibit the proliferation of MBP-specific T cells by 90%. The suppressor T cells obtained were weakly responsive to MBP and required the presence of the autologous PBMC for proliferation. Furthermore, proliferation of these suppressor T cell lines was restricted by HLA-DR molecules (for CD4+ Ts lines) and HLA class I (for a CD8+ Ts line). The suppressor T cell lines generated and the techniques described in this study may be helpful in our understanding of the events involved in the immune regulation in MS and other autoimmune diseases.


Analysis of the expression of CD5 by human B cells and correlation with functional activity.

B cells expressing the CD5 marker in the mouse have been suggested to be a separate lineage and a major source of autoantibody production. In man, this relationship is less clear. Studies were therefore undertaken to determine whether human CD5+ B cells represent a distinct lineage of cells that differ in patterns of antibody production from CD5- B cells. In normal B cell populations, CD5 was expressed by a mean of 24.0 +/- 2.8% (n = 10) of CD20+ B cells. Of note, an increased frequency of CD5+ B cells was not found in patients with systemic lupus erythematosus (mean of 17.9 +/- 2.8%, n = 16). Analyzing CD5+ B cells for cell membrane Ig isotype expression demonstrated similar frequencies of IgG and IgA expressing cells as were found on the CD5- B cell population, although the frequency of IgM+ cells was slightly increased. Incubation of CD20+ B cells with phorbol myristate acetate (PMA) for 72 hr increased the frequency of CD5 expressing B cells by more than threefold. CD5 expression was also increased by coculture with anti-CD3-activated T cells and most markedly by simultaneous stimulation with both PMA- and anti-CD3-activated T cells (greater than 50% positive). Analysis of CD5- B cells clearly indicated that stimulation with PMA or anti-CD3-activated T cells induced the majority to become CD5+ transiently. Functional analysis of Ig production by CD5+ and CD5- B cells stimulated with anti-CD3-activated T cells indicated that both populations in normals produced IgM and a variety of autoantibodies in comparable amounts, whereas the CD5- B cells produced greater quantities of IgG. B cells were activated with anti-CD3-stimulated T cells followed by separation into CD5+ and CD5- populations. The largest amount of Ig was produced by CD5- B cells that were induced to express CD5, although all populations produced some Ig. These data suggest that CD5 behaves as an activation marker on human B cells rather than as a marker for a distinct lineage of cells. Moreover, CD5 expression does not appear to identify a population of resting B cells with a greater capacity to produce antibodies to DNA or other autoantibodies.


B cell presentation of a tolerogenic signal to Th clones.

Lightly irradiated (950 R) splenic B cells were inefficient, in comparison to unseparated spleen cells, in stimulating antigen-specific proliferation of Th1 clones specific for human gamma globulin (HGG). This inefficiency was due to antigen-specific inactivation: Th1 clones preincubated with HGG and lightly irradiated B cells or mitomycin C-treated B cells were unable to proliferate to HGG in secondary cultures. In contrast to Th1 clones, Th2 clones proliferated well in response to B cell APC, and showed no decrease in their subsequent antigen-induced proliferative capacity after exposure to lightly irradiated B cells and HGG. However, preincubation of Th2 with lightly irradiated B cells and HGG did inactivate the capacity of Th2 to provide help for antibody production in secondary cultures. These results suggest that under certain conditions B cells may present antigen to Th1 and Th2 cells in a tolerogenic rather than an immunogenic manner.