Fragmentation of Therapeutic Human Immunoglobulin Preparations

Some commercial batches of human therapeutic immunoglobulins (Ig) have been found to show evidence of molecular fragmentation when examined by molecular sizing methodologies including sodium dodecyl sulphate polyacrylamide gel electrophoresis [SDS-PAGE] and size exclusion high performance liquid chromatography (SE-HPLC). These batches all demonstrated impaired immunobiological activity (efficacy) as assessed by Fc function measured using a rubella haemolytic assay and as such are likely to be subpotent for therapeutic use. Fragmented Igs were characterized by the presence of at least three protein bands and peaks additional to monomeric IgG. Incubation of Igs with blood enzymes (plasmin and kallikrein) reproduced the fragmentation patterns observed for intrinsically degraded batches, suggesting that fragmentation occurred by contamination with these proteases from the source material (human blood) during manufacture. Intravenous Igs (IVIG) were found to be more susceptible to proteolysis than intramuscular Igs, probably as a consequence of the post-fractionation processing that some IVIGs receive which may induce molecular alterations, allowing enzyme access and fragmentation. Two of the products examined were found to be relatively resistant to proteolysis and both were formulated by processes that limit enzyme activity. These processes were inclusion of an enzyme inhibitor, α₂-macroglobulin, and formulation at acidic pH. Enzyme carry-over into the final product is a likely cause of Ig fragmentation, and reduction in levels of such contamination should lead to improvements in product stability and efficacy.     

Isolation of Chlamydia trachomatis from eye secretion (tears).

Shedding of Chlamydia trachomatis in the eye secretion (tears) of patients with either hyperendemic trachoma or paratrachoma was studied. The method of collection of eye secretion with cellulose sponges is proved to be simple, faster, and more practicable and yielded a higher rate of chlamydial isolation than aspiration. The chlamydial isolation rates in eye secretion in chlamydia-positive paratrachoma patients in London or trachoma patients in Iran was 84 and 49% respectively. It was found that the chlamydial isolation rate from eye secretion is directly related to the number of inclusions present in the conjunctival swabbings. The results of this study indicated that patients with moderate to severe hyperendemic trachoma or paratrachoma are the main reservoir of infection. In the developing countries of the Middle East and Africa the shedding of chlamydia in the eye secretion of persons with these diseases is a major factor in the transmission of them by means of flies, fingers, towels, or bed clothes.     

"Doctor, my feet hurt"

Many patients complain of painful feet. While this problem may seem minor to the physician, it causes the patient considerable distress. Some of these conditions are readily amenable to treatment; others require continuing care. Much relief can be given by simple office procedures and by correcting ill-fitting footwear.     

Calibration and use of an in-house anti-measles IgG standard serum.

For the purpose of research a large quantity of anti-measles IgG working reference serum was needed. A pool of sera from five teenagers was prepared and named Alexandre Herculano (AH). In order to calibrate the AH serum, 18 EIA assays were performed testing in parallel AH and the 2nd International Standard 1990, Anti-Measles Antibody, 66/202 (IS) in a range of dilutions (from 1/50 to 1/25,600). A method which compared parallel lines resulting from the graphic representation of the results of laboratory tests was used to estimate the power of AH relative to IS. A computer programme written by one of the authors was used to analyze the data and make potency estimates. Another method of analysis was used, comparing logistic curves relating serum concentrations with optical density by EIA. For that purpose an existing computer programme (WRANL) was used. The potency of AH relative to IS, by either method, was estimated to be 2.4. As IS has 5000 milli international units (mIU) of anti-measles IgG per millilitre (ml), we concluded that AH has 12,000 mIU/ml.     

A proinflammatory role for IL-18 in rheumatoid arthritis

IL-18 is a novel cytokine with pleiotropic activities critical to the development of T-helper 1 (Th1) responses. We detected IL-18 mRNA and protein within rheumatoid arthritis (RA) synovial tissues in significantly higher levels than in osteoarthritis controls. Similarly, IL-18 receptor expression was detected on synovial lymphocytes and macrophages. Together with IL-12 or IL-15, IL-18 induced significant IFN-gamma production by synovial tissues in vitro. IL-18 independently promoted GM-CSF and nitric oxide production, and it induced significant TNF-alpha synthesis by CD14(+) macrophages in synovial cultures; the latter effect was potentiated by IL-12 or IL-15. TNF-alpha and IFN-gamma synthesis was suppressed by IL-10 and TGF-beta. IL-18 production in primary synovial cultures and purified synovial fibroblasts was, in turn, upregulated by TNF-alpha and IL-1beta, suggesting that monokine expression can feed back to promote Th1 cell development in synovial membrane. Finally, IL-18 administration to collagen/incomplete Freund's adjuvant-immunized DBA/1 mice facilitated the development of an erosive, inflammatory arthritis, suggesting that IL-18 can be proinflammatory in vivo. Together, these data indicate that synergistic combinations of IL-18, IL-12, and IL-15 may be of importance in sustaining both Th1 responses and monokine production in RA.     

A population-based study of prostate cancer chemotherapy

The use of chemotherapy in the treatment of prostate cancer is a relatively recent development, with no published data on the patterns of care in the UK. We carried out a population-based study to assess variation in the use of prostate cancer chemotherapy over time in a UK cancer network.     

Speckle Tracking Echocardiography in Pediatric and Congenital Heart Disease

Assessment of myocardial strain using speckle tracking echocardiography is an emerging echocardiographic technique that is increasingly used in the diagnosis and management of acquired heart disease in adults. In pediatric heart disease, this is still mainly considered as a research tool as the application of this technology has been slowed by the lack of vendor‐independent technology and of normative data across the different age ranges. We believe that the technology has potential applications for the early detection of myocardial dysfunction, the quantification of ventricular function in congenital heart disease, and the detection of dyssynchrony.     

Validation of a new highly standardised, lab-independent whole-blood leukocyte function assay for clinical trials (ILCS)

Physical stress induced in healthy volunteers by the Loughborough intermittent shuttle test (LIST) was used to validate a newly developed whole-blood cell culture system (Instant leukocyte culture system (ILCS). Exercise induced immune modulation was investigated through measurement of cytokine levels after activating leukocytes in peripheral blood ex vivo using the physiologic stimulant lipopolysaccharide (LPS). LPS induced production of three different cytokines, interferon gamma (IFNgamma), interleukin-6 (IL-6), and interleukin-10 (IL-10). IFNgamma levels were significantly decreased (P = 0.02 and P = 0.001 ) and IL-10 levels significantly increased (P= 0.04 and 0.03) after exercise. LPS induced IL-6 production was only marginally further increased by exercise. In conclusion, the ILCS system provided a reliable ex vivo method, showing common as well as subject specific features in the time course of the immune modulation caused by the LIST protocol. This system will be useful for studies of the elderly, where cytokine standardisation is notoriously difficult.     

Acute conjunctivitis caused by an atypical chlamydial strain: Chlamydia IOL 207.

A case of acute conjunctivitis in man caused by Chlamydia IOL 207 is described. Chlamydiae were isolated from the eye, and rising titres of type-specific antibodies to C. IOL 207, in serum and tears demonstrated an aetiological role for this agent. C. IOL 207 is an atypical chlamydial strain which serological studies suggest is widespread in some communities.