Biomedical subjects
N Young
Publications and source records attributed to N Young.
Effective planning for office and outpatient chemotherapy administration.
The trend in cancer care today is to successfully maintain the patient outside the traditional inpatient setting. This review has focused on some of the advantages and disadvantages in the administration of chemotherapy at home, the physician's office, and the ambulatory care facility. Effective planning for chemotherapy administration and the selection process for each of these settings depends on the needs of the patient and family. We can conclude that, although it is possible to use a combination of setting to maximize patient care, the ambulatory care concept provides broader choices for patients, families, and clinicians.
Viruses and bone marrow failure.
Some generalizations can be drawn from a review of virus-associated bone marrow failure. The story of B19 parvovirus illustrates that viral infection may be an occult cause of marrow failure. Although the epidemiology of transient aplastic crisis suggested a viral aetiology, the implication of a single virus was surprising; the sporadic appearance of chronic bone marrow failure in immunosuppressed persons has had none of the features of a viral illness. The incrimination of parvovirus in these cases required development of specific immunological and molecular assays. Human and animal retrovirus studies have shown that small changes in the virus genome can have dramatic effects on the biology of the infectious agent and its pathogenicity in infected hosts. In Epstein-Barr virus infection, the host's immune response may play a more important role in mediating disease than virus cytotoxicity. Finally, the association of aplastic anaemia with hepatitis may be underestimated because of the inability to diagnose virus infection without obvious liver disease. The true spectrum of bone marrow disease due to virus infection is not known.
Decreased interleukin 1 production in aplastic anaemia.
Interleukin 1 (IL-1) is an important regulator of immune system function. IL 1 also affects haematopoiesis in vitro: it causes release of colony stimulating factors from fibroblasts and endothelial cells and can directly act on primitive haematopoietic stem cells. We investigated IL 1 production in vitro by stimulated peripheral blood mononuclear cells of patients with aplastic anaemia (N = 17), patients with other haematologic diseases (N = 27), and normal individuals (N = 22) using a bioassay for IL 1 activity. Ten aplastic patients showed markedly decreased IL 1 production. IL 1 production by fibronectin-affinity purified monocytes was decreased in six of seven of these patients; in three other cases, in which IL 1 mononuclear cell production was undetectable, sufficient monocytes could not be isolated. IL 1 alpha and IL 1 beta precursor molecules were also absent or much decreased when mononuclear cell lysates from these patients were analysed by immunoblot using specific polyclonal sera. Aplastic patients with low IL 1 production were distinguished by the severity of their disease and the degree of neutropenia. Patients with myelodysplasia with comparable degrees of pancytopenia had normal IL 1 production. This is the first example of deficient haematopoietic growth factor production in a bone marrow failure syndrome. Decreased IL 1 production may contribute to the pathogenesis of some cases of aplastic anaemia and to susceptibility to infection.
Translational regulation of B19 parvovirus capsid protein production by multiple upstream AUG triplets.
The B19 parvovirus produces two capsid proteins in strikingly different quantities (VP1 less than 4%, VP2 greater than 96%) from overlapping RNAs that are derived from the same transcription unit. Immediately upstream from the VP1 translation initiation site is an unusual sequence containing multiple ATG triplets. During RNA processing this sequence is spliced out of VP2 RNA. To test the regulatory role on translation of this sequence containing upstream AUGs, synthetic RNAs were produced in vitro by T7 RNA polymerase from various plasmid constructions. Translation of VP1 RNA was very inefficient compared to VP2 RNA in a cell-free system, indicating that capsid protein production was regulated at the level of translation. Removal of upstream AUG sequences from VP1 RNA greatly increased the efficiency of translation. Conversely, the addition of the same AUG-rich sequence upstream of the initiation site of VP2 decreased its translation. These data indicate that an upstream AUG-rich region acts as a negative regulatory element in the translational control of B19 capsid protein production.
Carcinosarcoma of the uterine cervix initially interpreted as high grade sarcoma.
Carcinosarcoma of the uterine cervix is a rare tumor. A case of carcinosarcoma of uterine cervix initially interpreted as sarcoma, NOS, is presented along with immunohistochemical findings.
A comparison between the in vivo and in vitro activity of five potent and competitive NMDA antagonists.
1. Phosphonate analogues of glutamate have been tested and compared as N-methyl-D-aspartate (NMDA) antagonists in electrophysiological and binding experiments. The compounds tested were three established NMDA antagonists: D-2-amino-5-phosphonopentanoate (D-AP5), DL-2-amino-7-phosphonoheptanoate (DL-AP7), 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP), and two novel putative NMDA antagonists: 3-(2-carboxypiperidin-4-yl)propyl-1-phosphonate (CPPP) and 3-(2-carboxy-piperidin-4-yl)methyl-1-phosphonate (CPMP). 2. When administered electrophoretically to rat spinal neurones in vivo, these compounds were found to be selective NMDA antagonists with little effect on excitations evoked by quisqualate and kainate. CPMP and CPPP were approximately equipotent with CPP and about 5 times more potent than D-AP5. 3. Following systemic administration, 2-5 mg kg-1 i.v. of CPP, CPMP and CPPP reduced NMDA-evoked excitations by 70-100% whereas 50-100 mg kg-1 of D-AP5 and DL-AP7 produced a similar effect. The onset of the effects required 20-30 min and lasted more than six hours. 4. On bath application to cortical wedges, the IC50 values (microM) for antagonism of 40 microM NMDA were: CPP, 0.64 +/- 0.06 (mean +/- s.e.mean; n greater than 4); CPMP, 1.65 +/- 0.13; CPPP 0.89 +/- 0.09; D-AP5, 3.7 +/- 0.32; DL-AP7, 11.1 +/- 2.1; and DL-AP4 and DL-AP6 were inactive at 100 microM. 5. In binding studies with [3H]-CPP, the Ki values (nM) were: CPP, 446 + 150 (mean + s.e.mean; n > 3); CPMP, 183 + 74 and CPPP, 179 +/- 13 whereas against NMDA (lO microM)-stimulated [3H]- TCP (thienylcyclohexylpiperidine) binding the ICjo values (microM) for CPMP and CPPP respectively were 5.6 + 2.7 and 4.5 + 2.2. 6. Systemic administration of CPPP and CPMP, at doses sufficient to antagonize NMDA, also reduced cardiovascular responses to 5-hydroxytryptamine (Bezold-Jarisch reflex). This illustrates a role for NMDA receptors in central cardiovascular control. 7. The results indicate the systemic doses of piperidine and piperazine analogues of D-AP5 which may be used for assessing the role ofNMDA receptors in central synaptic function.
Functional mapping of the genome of the B19 (human) parvovirus by in vitro translation after negative hybrid selection.
We have analyzed the coding capacity of B19 parvovirus transcripts by in vitro translation using the negative hybrid selection technique. Five different antisense oligonucleotides (18-mers) corresponding to different portions of the B19 genome were hybridized to RNA samples extracted from human erythroid bone marrow cells infected with B19 parvovirus in vitro, and RNase H was added to cleave specific B19 RNA molecules at selected sites. B19-specific translation products of these RNA samples were determined by immunoprecipitation. We localized the B19 nonstructural protein to the left-side transcript and the two capsid proteins to overlapping transcripts from the right side of the genome.
The gene encoding the nonstructural protein of B19 (human) parvovirus may be lethal in transfected cells.
The B19 parvovirus is a cause of bone marrow failure in humans. B19 is toxic to erythroid progenitor cells in vitro. Viral products possibly responsible for toxicity were explored by transfection of cloned B19 genome into HeLa cells. The nonstructural (NS) protein was detected in cells 30 h after transfection. Plasmids containing the B19 genome were transfected with selectable marker genes in stable transformation assays. Plasmids that contained the left side of the B19 genome, which encodes the NS protein of the virus, inhibited antibiotic-resistant colony formation. Transformation occurred when NS protein expression was blocked by mutation. Suppression of transformation by NS protein was not tissue specific, suggesting a role for NS protein in toxicity for nonpermissive cells without parvovirus replication or virion accumulation.
Hematologic and hematopoietic consequences of B19 parvovirus infection.
In hybridization experiments, B19 shows some reactivity with autonomous rodent parvoviruses but none with adenoassociated virus sequences; its termini are more closely related to adenoassociated virus than to autonomous parvoviruses. B19 shares with all parvoviruses regions of conserved homology in the left side of the genome. The absence of an internal promoter and its unusual pattern of transcription sets B19 apart from both dependent and autonomous parvoviruses. Although clearly an autonomous parvovirus, in its extraordinary fastidious behavior B19 resembles a dependent parvovirus, capable of replication only in the special nuclear milieu of terminally differentiating erythroid cells. Adaptations at the molecular level may have been necessary for B19 parvovirus to acquire its high degree of specificity and low level of pathogenicity and thus succeed in human populations.
A multicenter trial of antithymocyte globulin in aplastic anemia and related diseases.
One hundred fifty patients with bone marrow failure were treated in three groups with antithymocyte globulin (ATG; Upjohn, Kalamazoo, MI) in a multicenter trial. Patients were assessed at 3, 6, and 12 months after initiation of treatment by three criteria: transfusion independence, clinical improvement, and blood counts. Group I consisted of 77 patients with acute severe aplastic anemia, randomized to receive either ten or 28 days of ATG. There was no significant difference between the two arms of this protocol: 47% of all patients were clinically improved and 31% were transfusion independent at 3 months. Of the severely affected patients, 27% died before 3 months; most deaths occurred early in treatment. Factors associated with survival in severely affected patients included male sex, age less than 40 years, absolute neutrophil count greater than 200/microL, and idiopathic etiology. Neutrophil counts generally increased by 8 weeks after treatment, but patients continued to show improvement to 1 year posttreatment. In Group II, 44 patients with moderate or chronic severe aplastic anemia were randomized to receive either ten days of ATG or 3 months of high-dose nandrolone decanoate. No patient initially treated with androgens recovered, but 28% of ATG-treated cases achieved transfusion independence at 3 months. Group III consisted of patients with a variety of bone marrow failure syndromes. Patients with pancytopenia and cellular bone marrow showed response rates similar to those of patients with chronic or moderate aplastic anemia.
Patients with bone marrow failure demonstrate decreased cutaneous reactivity to human C5a.
In vivo studies have shown that human C5a, a potent complement-derived anaphylatoxin and chemoattractant, produces immediate inflammatory reactions following intradermal injection in human skin. At concentrations within its potential physiologic range, intradermal injection of C5a elicits immediate wheal and flare reactions, increased vascular permeability, mast cell degranulation, and neutrophil-rich infiltrates. To assess the relative contribution of interacting cellular elements to C5a-induced inflammation in normal human skin, purified human C5a was tested intradermally in 8 patients with bone marrow failure (BMF). Reactions to C5a in patients with BMF were compared with responses at identical test sites in healthy volunteers and other patients with cutaneous disorders. Patients with BMF demonstrated significantly less wheal and flare reactivity following intradermal injection of C5a than controls (p less than 0.05 and less than 0.02, respectively). In these studies, patients with the greatest cytopenia generally showed the least cutaneous reactivity to human C5a. Biopsies of C5a test sites in patients with BMF revealed an absence of leukocytes in marked contrast to neutrophil-rich infiltrates observed at test sites in healthy volunteers. Avidin-fluorescent and/or Giemsa staining of skin biopsies revealed no difference between the number of dermal mast cells in patients with BMF and samples of normal human skin. In addition, skin test studies with histamine (2 micrograms) and morphine (5 micrograms) performed to assess cutaneous vascular and mast cell responsiveness in patients with BMF, normal volunteers and controls with rhinitis revealed no significant differences in cutaneous reactivity to these pharmacologic agents. These in vivo studies demonstrate that patients with BMF specifically exhibit decreased cutaneous reactivity to human C5a and suggest that neutrophils make an important and an immediate contribution to inflammatory responses elicited by this anaphylatoxin.
Lymphocyte phenotype and lymphokines following anti-thymocyte globulin therapy in patients with aplastic anaemia.
Twenty-two patients with adult onset aplastic anaemia were analysed before and after therapy with anti-thymocyte globulin (ATG). Lymphocyte phenotype, lymphokine levels or production, and haematopoietic progenitor cell number were measured 3 months after therapy; clinical response was determined 1 year post-therapy. By flow cytometry there was a significant reduction in both the proportion and absolute number of peripheral blood lymphocytes expressing activation antigen Tac (IL-2 receptor) and in the proportion of HLA-DR+ lymphocytes. For T cells bearing HLA-DR, there were proportional decreases in both activated helper and suppressor cells. There was no statistically significant difference pre-ATG to post-ATG in the absolute numbers of total, helper and suppressor lymphocytes. In all 10 haematologic responders the number of Tac bearing lymphocytes after ATG therapy was in the normal range, but half of 12 non-responding patients continued to have abnormally elevated numbers of Tac+ T cells. The proportion of Tac+ cells were not related to transfusion history. Gamma-interferon levels in serum by radioimmunoassay were elevated in almost half the aplastic patients; post-ATG, gamma-interferon was detectable in only three patients. Haematologic response to ATG therapy was associated with increased numbers of haematopoietic progenitors post-treatment, but pre-treatment values were not predictive of a response. These results are consistent with a pathogenic role for activated T-cells and their lymphokine products and suggest that the target of ATG therapy may be a Tac+ lymphocyte.
Neutropenia associated with large granular lymphocytes responsive to corticosteroids in vitro and in vivo.
We describe a patient with neutropenia associated with increased circulating large granular lymphocytes (LGL). Absolute neutropenia was accompanied by the absence of myeloid precursor cells in the bone marrow. No myeloid progenitor cells (CFU-C) could be detected by in vitro colony culture. The peripheral blood was also remarkable for the presence of a population of large granular lymphocytes demonstrable by conventional staining. These cells in flow microfluorometry studies expressed antigens Leu 4 (T-cell antigen receptor), Leu 7 (natural killer cell marker), Leu 2 (suppressor cell marker), and HLA-DR (activation marker); they lacked Leu 1 (a pan-T cell antigen), Leu 3 (helper cell marker) and Tac (interleukin 2 receptor). Hematopoietic colony formation in vitro improved with addition of corticosteroids to the culture medium or elimination of the LGL population with complement-mediated cytotoxicity. Anti-neutrophil antibodies were present prior to and following therapy. Clinically, administration of prednisone resulted in a normalization of the total white blood cell count and absolute polymorphonuclear cell number, an increase into the normal range of the number of CFU-C, and elimination of the LGL population. In this case of steroid-responsive LGL-associated neutropenia, laboratory studies suggested direct suppression of myelopoiesis by steroid-responsive LGL.
Novel transcription map for the B19 (human) pathogenic parvovirus.
The B19 parvovirus, a small single-stranded DNA virus of 5.4 kilobases, is pathogenic in humans. B19 has remarkable specificity for erythroid progenitor cells and has been propagated in vitro only with human erythroid bone marrow. Replication of viral DNA and the viral protein products of B19 appear similar to those of other animal parvoviruses. However, B19 transcription had unusual features in comparison with that in other animal parvoviruses. At least nine overlapping poly(A)+ transcripts were identified in infected cells; all but one contained large introns. B19 differed from other parvoviruses in the initiation of all transcripts at a strong left side promoter (p6) and the absence of a functional internal promoter; the presence of short 5' leader sequences of about 60 bases and very large introns for RNAs encoded by the right side of the genome; two separate transcription termination sites, in contrast to cotermination at the far right side of the genome for other parvoviruses; the probable utilization by three transcripts of a variant polyadenylation signal (ATTAAA or AATAAC) in the middle of the genome; and the abundance of two unique transcripts from the middle of the genome which did not code for capsid proteins. The unusual transcription map of B19 suggests that regulation of the relative abundance of transcripts occurs by splicing and termination-polyadenylation events rather than by promoter strength. In combination with the published nucleotide sequence, the novel transcription map separated the pathogenic B19 virus at a molecular level from other animal parvoviruses and human adeno-associated virus.
Characterization of capsid and noncapsid proteins of B19 parvovirus propagated in human erythroid bone marrow cell cultures.
The major capsid and noncapsid proteins of the pathogenic parvovirus B19, propagated in vitro, were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoprecipitation, and immunoblot of the erythroid fraction of infected human bone marrow cell cultures. There were two capsid proteins of 58 kilodaltons (kDa; the major species) and 84 kDa (the minor species). Newly synthesized capsid viral proteins were present in the supernatants of infected cultures. The major noncapsid protein of 77 kDa was localized to the nucleus.
Multiple sclerosis and the workplace: report of an industry-based cluster.
Eleven cases of MS occurred within a 10-year period in a zinc-related manufacturing plant. The observed disease incidence was greater than expected from population data, using multiple approaches to statistical analysis (p less than or equal to 0.01). A case-control study, performed to examine several zinc parameters in blood, failed to indicate specific abnormalities among the MS patients, but all subjects (both MS and controls) working in the plant demonstrated higher serum zinc levels than all subjects (MS and controls) not working there.
Tobacco mosaic virus replicase and replicative structures.
The RNA-dependent RNA polymerase (replicase) mediating the replication of tobacco mosaic virus (TMV) has been investigated in a number of laboratories over a period of 20 years. Cell-free enzyme preparations have been prepared which can continue the synthesis of nascent complementary RNA, initiated in vivo; however, the enzyme does not require, nor does it respond to, exogenous viral RNA as a template. The presence in plants of a virus-stimulated, host-encoded RNA-dependent RNA polymerase (RdRp) has added confusion to this field; it is now generally conceded, however, that this enzyme is not the TMV replicase. Our recent studies have emphasized several aspects of TMV RNA replication. We have examined the nature of TMV replicative structures synthesized in vitro by utilizing a partially purified enzyme preparation isolated from TMV-infected tobacco tissue. Radiolabelled products of the reaction were analysed on agarose gels and fractions with the predicted electrophoretic migration and nuclease sensitivities of replicative form (RF) and replicative intermediate (RI) were isolated. These fractions were hybridized to a collection of bacteriophage M13 clones containing portions of the TMV genome of both plus and minus polarity. The nascent synthesis in the RI-like molecules was restricted to the plus viral strand, while the new synthesis in the RF-like molecules was of both plus and minus polarity. Solubilization of the membrane-bound replicase with the non-ionic detergent CHAPS has yielded complexes which remain in solution after high-speed centrifugation. The solubilized replication complexes have been utilized as starting material for enzyme purification by Sepharose 4B gel filtration chromatography. The intracellular site of synthesis of TMV RNA has been reinvestigated in the light of reports suggesting a nuclear site of replication. The conclusion for nuclear synthesis has been based on fractionation of subcellular homogenates of virus-infected leaves or mesophyll protoplasts and identification of virus-related proteins associated with these fractions. In our studies, however, we conclude that these procedures can be misleading in that the 126,000 Mr TMV protein (and replicase activity) were found in all fractions of the homogenate analysed. Double-stranded TMV RNA, on the other hand, was barely detectable in preparations of purified nuclei; instead it was concentrated in the post-nuclear supernatant, suggesting that the nucleus is not the site of TMV RNA synthesis.