Biomedical subjects
Publications and source records attributed to D R Shaw.
Fatal severe rhabdomyolysis and anuric renal failure developed in a young man who had contracted hand, foot and mouth disease during an epidemic of this disease. Viral studies implicated coxsackievirus A16 as the infecting agent. This appears to be the first reported case of rhabdomyolysis that was associated with hand, foot and mouth disease and infection with coxsackievirus A16.
In a one-year prospective study of 106 adults (mean age, 60 years) who were admitted to hospital with community-acquired pneumonia, an aetiological diagnosis was made in 82 (77%) patients. Streptococcus pneumoniae was considered to be responsible for 44 (42%) and respiratory viruses for 19 (18%) infections. Other aetiological agents that were found in a smaller number of patients included Haemophilus influenzae (9% of patients), enteric Gram-negative bacilli (8% of patients), Staphylococcus aureus (3% of patients), Legionella spp. (3% of patients), Mycobacterium tuberculosis (3% of patients), Mycoplasma pneumoniae (8% of patients) and Chlamydia psittaci (5% of patients). The mortality was 10% and was related significantly to increasing age and to coexisting heart and lung disease. Antibiotic treatment that was commenced before admission to hospital and investigations were undertaken reduced significantly the isolation rate of susceptible bacterial pathogens. The Gram-stained smear of sputum was valuable in establishing a tentative diagnosis of bacterial pneumonia. The most-useful tests in making an early diagnosis proved to be those which detected pneumococcal and mycoplasmal antigens, blood cultures and culture of sputum for appropriate bacterial pathogens.
Dictyostelium discoideum ubiquitin mRNAs are regulated in a complex fashion during spore germination and multicellular development. Species of mRNA of 1900, 1400, 1100, 840, 580, and 500 nucleotides (nt) are found which are expressed differentially during different stages of development. DNA blot analysis indicates that ubiquitin genes constitute a multigene family of at least six genes. cDNAs representing all the ubiquitin mRNA transcripts were isolated and sequenced. The Dictyostelium mRNAs are organized as tandem repeats of the 76 amino acid ubiquitin unit (228 nt). We isolated one cDNA containing seven of these tandem repeats, and two different five- and three-repeat cDNAs. In addition, 2 cDNAs containing a single ubiquitin repeat fused at its 3' end to an unrelated 52 and 78 amino acid extension were identified. There is a remarkable similarity in the sequences of the non-ubiquitin extensions among yeast and mammalian counterparts. The extensions are very basic, containing approximately 30% lysine/arginine. Another common feature of these proteins is the presence of a common structural motif containing cysteine residues at conserved positions, suggesting a metal binding domain that matches a consensus sequence of Xenopus transcription factor TFIIIA and other nucleic acid binding proteins. The characterization of ubiquitin cDNAs and genomic sequences in D. discoideum now makes the understanding of its developmental regulation feasible.
Ten patients with idiopathic thrombocytopenic purpura (ITP) were studied before and following a rise in circulating platelets subsequent to infusions of intravenous gammaglobulin (400 mg/kg/day x 5 days). We quantitated the amount of circulating IgG capable of binding to normal donor platelets in vitro using an 125I-monoclonal anti-human IgG assay, as well as the amount of IgG associated with the patients' platelets before and following therapy. We found no evidence for a decrease in platelet-specific IgG antibodies in these patients undergoing an acute response to therapy. These data suggest that the short-term efficacy of intravenous gammaglobulin is due to effects other than a substantive reduction in platelet reactive antibodies, such as the alteration of IgG-coated platelet destruction.
A Dictyostelium discoideum repetitive element composed of long repeats of the codon (AAC) is found in developmentally regulated transcripts. The concentration of (AAC) sequences is low in mRNA from dormant spores and growing cells and increases markedly during spore germination and multicellular development. The sequence hybridizes to many different sized Dictyostelium DNA restriction fragments indicating that it is scattered throughout the genome. Four cDNA clones isolated contain (AAC) sequences in the deduced coding region. Interestingly, the (AAC)-rich sequences are present in all three reading frames in the deduced proteins, i.e., AAC (asparagine), ACA (threonine) and CAA (glutamine). Three of the clones contain only one of these in-frame so that the individual proteins carry either asparagine, threonine, or glutamine clusters, not mixtures. However, one clone is both glutamine- and asparagine-rich. The (AAC) portion of the transcripts are reiterated 300 times in the haploid genome while the other portions of the cDNAs represent single copy genes, whose sequences show no similarity other than the (AAC) repeats. The repeated sequence is similar to the opa or M sequence found in Drosophila melanogaster notch and homeo box genes and in fly developmentally regulated transcripts. The transcripts are present on polysomes suggesting that they are translated. Although the function of these repeats is unknown, long amino acid repeats are a characteristic feature of extracellular proteins of lower eukaryotes.
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Variable region genes from mouse monoclonal antibody 17-1A (gamma 2a kappa) with specificity for human gastrointestinal malignancies have been paired with human immunoglobulin constant region genes (for heavy and light chains) to produce mouse/human chimeric immunoglobulin molecules (chIgG) for each of the four human IgG subclasses. Mouse 17-1A and the four chIgG bound similarly to two human colon cancer cell lines and had comparable binding affinities. The chIgG1 and chIgG3 molecules mediated lymphocyte and monocyte antibody-dependent cell-mediated cytotoxicity (ADCC) to colon cancer tumor cell lines comparable to that of the parent murine 17-1A. The chIgG2 and chIgG4 molecules were able to mediate ADCC to colon cancer cell lines but were clearly inferior to the chIgG1 and chIgG3 reagents. None of the chIgG antibodies or the murine 17-1A was able to mediate complement lysis of colon cancer cell lines. These studies demonstrate the ability to produce all four human IgG subclass chimeric molecules which retain biologic activity. We have confirmed the subclass preferences of human lymphocyte and monocyte Fc receptors for human IgG subclasses previously determined by studies with monomeric or aggregated IgG. These data may aid in the selection of chimeric antibodies for in vivo trials.
Mycoplasma hominis was isolated from the surgical wounds of two patients. One of these patients developed a septicaemia-like illness after coronary artery vein-graft surgery. Mycopl. hominis was isolated from the sternotomy wound and pleural fluid of this patient but not from his blood. No other pathogens were isolated. The second patient developed a low-grade soft-tissue infection due to Mycopl. hominis in a craniotomy wound; his condition responded to tetracycline treatment. The source of infection in both cases presumably was mycoplasmaemia which was secondary to urethral catheterization. Infections due to Mycopl. hominis are overlooked easily unless appropriate steps are taken to isolate and to identify these organisms.
Similar to other stages in Dictyostelium development, spore germination is a particularly suitable model for studying the regulation of gene expression, because developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. Spores are constitutively dormant and must be activated to germinate. Under the proper environmental conditions, spores germinate in a highly synchronous manner to give rise to individual amoebae that can then enter the vegetative growth phase. Protein synthesis is developmentally regulated during this process. Because protein synthesis is transcriptionally controlled during spore germination, the respective genes must be developmentally transcribed, and these can be isolated and analyzed. Three cDNA clones specific for mRNA developmentally regulated during spore germination have been characterized and used as probes to study mRNA accumulation and decay during spore germination. Because we are interested in defining the sequences of developmentally regulated genes that may relate to their regulation of transcription, we have sequenced the cDNAs and have isolated and sequenced their respective genomic clones. The sequences of the three gene families, their genomic organization, and their special structural features are described.
The mouse monoclonal antibody 17-1A (gamma 2a, kappa) recognizes a tumor-associated antigen expressed on human gastrointestinal malignancies and has been used in Phase I and II clinical trials. Chimeric genetic constructs have been produced using 17-1A variable region genes (VL and VH) and the constant region genes for human kappa light chains and gamma 1 heavy chains (C kappa and gamma 1). The chimeric gene constructs were transfected into mouse myeloma cells for antibody production. The secreted mouse/human chimeric antibody contains the antigen-binding domain of 17-1A and the human (gamma 1, kappa) constant regions. Native mouse 17-1A and the chimeric antibody (chIgG1) were analyzed for binding to two human colon cancer cell lines and for the mediation of cancer cell line antibody-dependent cellular cytotoxicity by normal human peripheral blood lymphocytes and monocytes in 4 h 51Cr-release assays. The 17-1A and chIgG1 gave similar results in these in vitro biologic assays. This study demonstrates the feasibility of using mouse/human chimeric antibodies in human therapeutic applications.
Mouse monoclonal antibody 17-1A is specific for an antigen expressed on cells of human gastrointestinal malignancies and has been used in radioimmune imaging and therapy trials for patients with colon and pancreatic cancer. The cell line SG3/5 was generated by transfection of a nonproducing mouse myeloma line (SP2/0) with a chimeric gene construct composed of variable regions from the mouse 17-1A immunoglobulin (gamma 2a, kappa) and constant regions of human k and gamma 3 immunoglobulin genes. The secreted immunoglobulin was bound by mouse monoclonal antibodies to human IgG(Fc) and IgG3 but not by staphylococcal protein A. Gel filtration HPLC profiles of purified chimeric antibody were similar to normal human IgG3 but quite different from native 17-1A and normal human IgG1, 2, and 4. Native and chimeric 17-1A had similar patterns of reactivity with colon cancer, other adenocarcinoma, and leukemic cell lines. Competitive inhibition documented that native and chimeric 17-1A had identical capacities to inhibit radiolabeled native 17-1A binding to colon cancer cell lines. Thus, the chimeric 17-1A exhibits molecular characteristics of normal human IgG3 but retains the specificity and binding affinity of the native 17-1A murine monoclonal antibody. The native and chimeric 17-1A mediated similar modest degrees of human lymphocyte and monocyte ADCC in a 4-hr 51Cr release assay, and both failed to mediate complement lysis of colon carcinoma cell lines in the presence of human complement. This human/mouse chimeric monoclonal antibody may be a good candidate for use in clinical trials because it retains the tumor antigen specificity and human effector cell recognition of the native 17-1A, would presumably have a fivefold to 10-fold longer circulating half-life in man, and should be considerably less immunogenic as compared with native murine immunoglobulins.
An 80-year-old woman developed acute hepatitis following her first exposure to nifedipine. This adverse effect was characterised by fever, chills, anorexia, nausea, liver tenderness, hepatitic liver function tests and peripheral blood eosinophilia. On liver biopsy the portal tracts were expanded with a mixed inflammatory cell infiltrate rich in eosinophils. The potential for the occurrence of this adverse effect must increase with the current expansion of indications for the use of nifedipine.
Spore germination in Dictyostelium discoideum is a particularly suitable model for studying the regulation of gene expression, since developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. The previous isolation of three cDNA clones specific for mRNA developmentally regulated during spore germination allowed for the quantitation of the specific mRNAs during this process. The three mRNAs specific to clones pLK109, pLK229, and pRK270 have half-lives much shorter (minutes) than those of constitutive mRNAs (hours). Using spore germination as a model, we studied the roles of ribosome-mRNA interactions and protein synthesis in mRNA degradation by using antibiotics that inhibit specific reactions in protein biosynthesis. Cycloheximide inhibits the elongation step of protein synthesis. Polysomes accumulate in inhibited cells because ribosomes do not terminate normally and new ribosomes enter the polysome, eventually saturating the mRNA. Pactamycin inhibits initiation, and consequently polysomes break down in the presence of this drug. Under this condition, the mRNA is essentially free of ribosomes. pLK109, pLK229, and pRK270 mRNAs were stabilized in the presence of cycloheximide, but pactamycin had no effect on their normal decay. Since it seems likely that stability of mRNA reflects the availability of sites for inactivation by nucleases, it follows that in the presence of cycloheximide, these sites are protected, presumably by occupancy by ribosomes. No ribosomes are bound to mRNA in the presence of pactamycin, and therefore mRNA degrades at about the normal rate. The data further indicate that a labile protein is probably not involved in mRNA decay or stabilization, since protein synthesis is inhibited equally by both antibiotics. We conclude that it may be important to use more than one type of protein synthesis inhibitor to evaluate whether protein synthesis is required for mRNA decay. The effect of protein synthesis inhibition on mRNA synthesis and accumulation was also studied. mRNA synthesis continues in the presence of inhibitors, albeit at a diminished rate relative to that of the uninhibited control.
We quantitated the amount of platelet surface-bound IgG using an 125I monoclonal anti-IgG assay in 149 patients with thrombocytopenia and 260 normal donors. The normal subjects had 122 +/- 5 molecules of IgG/platelet (mean +/- SE). Fifty-five patients with nonimmune thrombocytopenia had 338 +/- 37 molecules of IgG/platelet, whereas 67 patients with immune thrombocytopenia studied at the time of their initial evaluation had 4,120 +/- 494 molecules of IgG/platelet. An analysis of the distribution of values in these two groups indicated that 90% of the patients with immune thrombocytopenia had greater than 800 molecules of IgG/platelet, whereas only 7% of patients with nonimmune thrombocytopenia exceeded this amount. The immune thrombocytopenia patients included 39 idiopathic, 14 secondary, and 14 drug-induced disorders, and they did not significantly differ in their distribution of values for platelet IgG. The nonimmune thrombocytopenic patients included 12 cases with a platelet destructive mechanism; their platelet-bound IgG was similar to that of the other nonimmune patients. Twenty-seven patients with treatment resistant immune thrombocytopenia were also studied they had 2,100 +/- 670 molecules of IgG/platelet. Their values were significantly greater than those of the nonimmune thrombocytopenic patients and not significantly different from those of immune thrombocytopenic group. Their distribution of values was much broader, however, with 33% of patients having less than 800 molecules of IgG/platelet, suggesting possible alternate mechanisms in their thrombocytopenia. Thus, patients with immune thrombocytopenia have a high frequency of elevated IgG on the platelet surface which reflects the pathophysiology of this disorder. Quantitation of platelet-bound IgG provides a useful laboratory tool in the differential between immune and nonimmune thrombocytopenia.
During studies aimed at isolating myosin-specific genomic clones in Dictyostelium, we probed a lambda genomic library with a chicken myosin light-chain sequence (pML10). Many lambda recombinant Dictyostelium clones hybridized to the pML10 cDNA insert, indicating that this sequence was reiterated in the Dictyostelium genome. It was found that the 3'-noncoding region (pML10-NC) alone was responsible for these results. Dictyostelium DNA contained approximately 65 copies of a sequence(s) similar but not identical to that of pML10-NC. Southern blot analysis showed that pML10-NC hybridized to many Dictyostelium genomic DNA fragments of varying sizes generated by digestion with EcoRI, HindIII, or AluI. In addition, each of the Dictyostelium clones was different in its size, restriction map, and flanking sequences. It seems likely, therefore, that the sequences which hybridized to pML10-NC are scattered throughout the Dictyostelium genome and similar but not identical to each other or to pML10-NC. Thus, probing with pML10-NC has allowed us to select a family of closely related but not identical sequences. These D. discoideum sequences are not found in other slime mold species. No RNA complementary to pML10-NC was found in vegetative cells, 18 h culmination stage, spores, or 1- and 2-h germinating spores. pML10-NC-related sequences were present in two other Dictyostelium species but were absent in the related genus Polysphondylium.
The ability of several immunologic ligands to mediate extracellular cytolysis of sheep erythrocytes (E) by mouse macrophages was studied. E coated with rabbit IgG (EIgG), but not nonsensitized E or E coated with rabbit IgM (EIgM), were lysed and phagocytized by resident peritoneal macrophages as well as by macrophages activated in vivo with either BCG or Corynebacterium parvum or in vitro with lymphokine. EIgM incubated with mouse serum to deposit the third component of complement (C3) onto E (EIgMC) were ingested by thioglycolate-elicited and lymphokine-treated macrophages, but not by the other macrophage populations examined. However, none of the macrophages performed lysis of EIgMC, suggesting that engagement of macrophage C3 receptors by target cell-bound C3 was not a sufficient trigger for cytolysis. Lysis of E coated with both IgG and C3 (EIgGC) was moderately elevated over that of EIgG; this enhancement was not abolished after proteolytic destruction of the macrophage C3-rosetting capacity, indicating that C3 receptors were not responsible. EIgGC and EIgMC were more susceptible to hypotonic lysis than were either E, EIgG, or EIgM, suggesting that enhanced lysis of serum-treated E may be partially explained by increased E fragility.
We have utilized 'lysis from without' mediated by UV-inactivated bacteriophage T6 to eliminate extracellular bacteria in experiments measuring the internalization, intracellular survival and replication of Yersinia pestis within mouse peritoneal macrophages and of Shigella flexneri within a human intestinal epithelial cell line. The technique we describe has the following characteristics: (a) bacterial killing is complete within 15 min at 37 degrees C, with a greater than 10(3)-fold reduction in colony-forming units (CFU); (b) bacteria within cultured mammalian cells are protected from killing by UV-inactivated T6; (c) the mammalian cells are not observably affected by exposure to UV-inactivated T6. This technique has several advantages over the use of antibiotics to eliminate extracellular bacteria and is potentially widely applicable in studies of the interactions between pathogenic bacteria and host phagocytic cells as well as other target tissues.
One thousand five hundred serum drug levels lying outside the defined therapeutic range have been followed up by the clinical pharmacology service at the Royal Adelaide Hospital over a thirteen month period. It was found that digoxin, phenytoin and theophylline were the most frequently monitored drugs. Blood levels outside the therapeutic range tended to be on the high side with digoxin, but were usually sub-therapeutic for the other two. Drug related toxicity was observed in 11.2 percent of those with a high serum level. Several potential drug interacting situations were also noted during the study. The value of therapeutic drug monitoring, to maximise efficacy of therapy and minimise side effects, is stressed. However, correct blood sampling, based on a knowledge of the clinical pharmacology and pharmacokinetics of the drug, must be performed in order to obtain optimal benefit from this exercise.