Search PubMed⌕ Search

Biomedical subjects

M Little

Publications and source records attributed to M Little.

At least 199 records · Page 11Linked to original sources
PubMedDate not supplied

Regulated secretion and purification of recombinant antibodies in E. coli.

A plasmid for optimized protein expression of recombinant Fv antibodies (pOPE) in E. coli was used to express the variable domains of the murine monoclonal antibody HD39 specific for the human B-cell surface antigen CD22. The production of Fv antibodies by pOPE can be regulated over a wide range by varying the IPTG concentration. Antibodies that can discriminate between secreted and nonsecreted Fv antibody fragments were used to show that secretion is the limiting step for the production of functional Fv antibodies. IPTG concentrations above 20 microM increased the total antibody production, but did not yield larger amounts of secreted Fv antibodies. The addition of five histidines to the C terminus facilitates an easy single-step enrichment procedure based on immobilized metal affinity chromatography.

Amino Acid Sequence↗
PubMedDate not supplied

Recombinant human monoclonal antibodies. Basic principles of the immune system transferred to E. coli.

To produce human monoclonal antibodies in bacteria, a gene repertoire of IgM variable regions was isolated from human peripheral B lymphocytes by the polymerase chain reaction. Alternatively, synthetic antibody genes with random hypervariable regions are being generated that may provide libraries of even higher complexity. For the selection of specific monoclonal antibodies from these libraries, we have developed two E. coli vector systems that facilitate the surface display of an antibody physically linked to its own gene. The phagemid pSEX encodes a fusion protein of an antigen binding domain (Fv-antibody) with the docking protein (pIII) of filamentous phages. Specific antibody genes can therefore be enriched by antigen affinity chromatography. The plasmid pAP1 encodes a fusion protein of an Fv-antibody with a bacterial cell-wall protein. Bacteria carrying this plasmid express functional Fv-antibodies tightly bound to their surface. This should enable the selection of single cells with a fluorescence-assisted cell sorter (FACS) using labeled antigen or by adsorption to immobilized antigen. These vectors permit three major principles of the antibody response to be mimicked in E. coli: 1. Generation of a highly complex antibody repertoire; 2. Clonal selection procedures for library screening; and 3. The possibility of increasing a given affinity by repeated rounds of mutation and selection.

Antibodies, Monoclonal↗
PubMedDate not supplied

Idiotypic vaccine for treatment of human B-cell lymphoma. Construction of IgG variable regions from single malignant B cells.

Immunoglobulin idiotypes (Id) of malignant B cells represent highly specific markers which can be used for vaccination. PCR-amplification of immunoglobulin genes enables the rapid production of large amounts of Id vaccines. However, the separate amplification and subsequent recombination of heavy and light chains can lead to a loss of the relevant Id. To preserve the original chain pairs, we used single malignant B cells derived from an immunocytoma patient. Cytoplasm was extracted and the mRNA transcribed into cDNA. The VH and VL genes were then amplified by PCR and cloned into a vector for expression in E. coli. Id production was checked using an anti-Id mouse monoclonal Ab raised against the patient's tumor-specific IgG. One out of 3 constructs expressed the relevant Id. Analysis of the first 31 light chain residues revealed an identical sequence for the malignant B cells' IgG and the recombinant Id construct. Exchange of either the heavy or light chain with an unrelated chain resulted in loss of the Id. An unrelated sequence derived from the c-myc protein is coupled to the Id vaccine. The lymphoma patient was shown to have Abs to the c-myc sequence. This sequence therefore, increases the Id+ Ab's antigenicity. CD spectroscopy showed an alpha-helical structure for the c-myc epitope. In conclusion, a B-cell lymphoma autovaccine was produced containing immunogenic sequences that do not alter the steric conformation of the tumor-specific Id.

Animals↗
PubMedDate not supplied

A year's experience of Irukandji envenomation in far north Queensland.

Envenomation by the Irukandji jellyfish (Carukia barnesi) can result in an array of systemic symptoms known as Irukandji syndrome. In 1996, 62 people presented to Cairns emergency departments with Irukandji envenomation: 57 developed systemic symptoms, and 38 required parenteral narcotics. All patients were discharged home within 24.5 hours, except for two who required high-dependence care for pulmonary oedema. Patients were more likely to be stung on hotter days, with lower-than-average rainfall in the past seven days, and with winds from the north, but less-than-average wind speed. We offer a protocol for treating patients with Irukandji envenomation.

Adolescent↗
PubMedDate not supplied

Pressure immobilisation bandages in first-aid treatment of jellyfish envenomation: current recommendations reconsidered.

OBJECTIVE: To evaluate whether applying pressure equivalent to that of pressure immobilisation bandages (PIB) causes release of additional venom from discharged jellyfish nematocysts. DESIGN: In-vitro experiment--the venom beads released from electrically activated Chiropsalmus sp. nematocysts were viewed under direct microscopy before and after applying 40 mmHg pressure (replicating the pressure of PIB); and saline washings of discharged nematocysts before and after applying pressure were tested for toxicity (time to ventricular standstill after injecting into live prawns). RESULTS: Applying 40 mmHg pressure caused the venom beads to visibly increase in size, consistent with pressure expressing further venom from the discharged nematocysts. First washings of the nematocyst shafts before compression produced ventricular standstill in prawns within 60 seconds (n=3); second washings did not produce standstill during 540 seconds of observation (n=3); and washings after applying 40 mmHg pressure showed a return of toxicity, with ventricular standstill in all prawns within 180 seconds (n=3). CONCLUSION: Discharged nematocysts are by no means empty and harmless. Applying pressure results in further release of nematocyst venom. The currently recommended practice of applying PIB in the initial treatment of patients stung by a jellyfish may exacerbate the envenomation, and thus should not be recommended.

Animals↗
PubMedDate not supplied

Evaluating student experiences in a family medicine clerkship.

One hundred students on an eight-week family practice clerkship completed pre- and post-evaluations noting their experience and confidence in diagnosing and treating 20 common medical problems and doing 15 common tasks. Students' confidence increased significantly during the clerkship in dealing with 18 of 20 common problems and 13 of 15 common tasks. Confidence ratings were highly correlated with experiences on the clerkship. Even though clerkship experiences varied at different sites, students reported similar increases in confidence at all sites. A self-administered questionnaire is a simple and effective way of documenting student experience, comparing clerkship sites, and measuring achievement of course objectives.

Attitude of Health Personnel↗