Nocturnal haemoglobin oxygen desaturation in urban and rural East African paediatric cohorts with and without sickle cell anaemia: a cross-sectional study.

Low haemoglobin oxygen saturation (SpO2) predicts complications in children with sickle cell anaemia (SCA) in the North but there are few data from Africa, where the majority of the patients reside. We measured daytime and overnight SpO2 in children with SCA in routine follow-up clinic, and controls without symptoms of SCA, comparing rural (Kilifi, Kenya) and urban (Dar-es-Salaam, Tanzania) cohorts. Daytime SpO2 was lower in 65 Tanzanian children with SCA (TS; median 97 (IQR 94-100)%); p<0.0001) than in 113 Kenyan children with SCA (KS; 99 (98-100)%) and 20 Tanzanian controls (TC; 100 (98-100)%). Compared with 95 Kenyan children with SCA, in 54 Tanzanian children with SCA and 19 TC who returned for overnight oximetry, mean (KS 99.0 (96.7-99.8)%; TS 97.9 (95.4-99.3)%; TC 98.4 (97.5-99.1)%; p=0.01) and minimum nocturnal SpO2 (92 (86-95)%; 87 (78.5-91)%; 90 (83.5-93)% p=0.0001) were lower. The difference between children with SCA persisted after adjustment for haemoglobin (p=0.004). Urban Tanzanian children, with and without SCA, experience greater exposure to low daytime and night-time SpO2 compared with rural Kenyan children with SCA. Possible explanations include differences in the prevalence of obstructive sleep apnoea or asthma, alterations in the oxyhaemoglobin desaturation curve or cardiovascular compromise, for example, to shunting at atrial or pulmonary level secondary to increased pulmonary artery pressure. The fact that non-SCA siblings in the urban area are also affected suggests that environmental exposures, for example, air pollution, nutrition or physical exercise, may play a role. Further studies should determine aetiology and clinical relevance for the SCA phenotype in children resident in Africa.

Development of improved SNAP25 endopeptidase immuno-assays for botulinum type A and E toxins.

Botulinum neurotoxins contain proteases that cleave specific intra-neural proteins essential for neurotransmitter release. Toxin types A, E and C1 intra-cellularly cleave SNAP25 resulting in a flaccid paralysis. As a consequence, various different endopeptidase assays have been developed to specifically detect the toxins enzymatic activity, however, many of these suffer from variability, low sensitivity or unwanted interference exerted by product specific excipients. The current studies utilised solid phase synthesized SNAP25(137-206) peptide substrate, and specific antibody to either the SNAP25(190-197) or (173-180) octapeptide epitopes that become exposed following cleavage by toxin types A or E respectively. Assay sensitivity was increased 50 fold by the use of an optimal 0.5% Tween 20 concentration in tandem to 0.1% albumin together with an improved, simplified assay design without a pre-activation / reduction step. Sensitivities capable of detecting 0.01 LD50/ml (40fg/ml or 0.3fM) of type A toxin was achieved with a linear dose response between 0.1 and 1 LD50/ml. This provides sufficient sensitivity and precision (inter assay GCV of < 2%) for monitoring activity within any current or newly marketed therapeutic products containing less units per vial and may also make it applicable for other applications. Both purified haemagglutinin free and complexed toxins could be detected equally. Unlike type A, type E activity could unexpectedly be detected in the complete absence of reducing conditions and the optimal assay had a limit of detection of 0.2LD50/ml (4.8pg/ml) with a linear dose response between 1 and 10LD50/ml. The principle of using a detecting antibody to a substrate sequence buried within the native substrates alpha-helix may be further expanded to other specific enzyme cleavage reactions in the future.

Application of an in vitro endopeptidase assay for detection of residual toxin activity in tetanus toxoids.

Tetanus vaccine is composed of chemically denatured tetanus toxin (TeNT), thus safety testing requires confirmation of freedom from residual and reversible toxicity. Currently, TeNT activity is estimated using in vivo assay models. Information that TeNT acts by selectively inactivating protein leading to the blocking of release of neurotransmitters has provided the opportunity to develop in vitro biochemical assay for toxin activity. In this study we describe development and use of an in vitro endopeptidase assay for detection of TeNT activity in toxoid vaccine formulations.

Therapeutic botulinum type A toxin: factors affecting potency.

The type A neurotoxin produced by Clostridium botulinum is a potent neuromuscular blocking agent which causes paralysis by preventing the release of neurotransmitter from motor neurons. This property has resulted in the use of the toxin in the treatment of a number of neuromuscular diseases involving muscle spasms. At present, the only recognised assay to estimate accurately the potency of botulinum toxin in clinical preparations is bioassay, in which lethality is used as the endpoint. Such bioassay is inherently variable and large interlaboratory variability has been reported, highlighting problems for standardisation of activity in the absence of any commonly used reference preparation. In the present study, we have confirmed that many different assay conditions can affect potency estimates of clinical formulations of type A botulinum toxin. Further, our studies indicate that different preparations, because of their unique formulation and stability, are differentially affected by some of these assay conditions and that these differences might well contribute to the differences observed in their clinical use.

Refinement and validation of an alternative bioassay for potency testing of therapeutic botulinum type A toxin.

The type A neurotoxin produced by Clostridium botulinum is a potent neuromuscular blocking agent which causes paralysis by preventing the release of neurotransmitter from motor neurones. This property has led to the use of the toxin in the treatment of a number of neuromuscular diseases involving muscle spasms. At present, the only recognised assay with the specificity and sensitivity to estimate accurately the potency of botulinum toxin in clinical preparations is bioassay, in which lethality is used as the end point. Refinement of this assay, with respect to the end point, was explored on the basis of the development of flaccid paralysis of muscles following subcutaneous injection of the toxin at the inguinocrural region. Potency estimates, relative to in house reference preparations, for different therapeutic preparations obtained using flaccid paralysis as a scored response gave excellent agreement with estimates obtained in independent assay using the currently required control method. This study demonstrates that an alternative, more humane bioassay for potency testing of clostridia neurotoxins gives valid estimates equivalent to those currently in use.

Immunological detection of Clostridium botulinum toxin type A in therapeutic preparations.

The potent neurotoxins produced by strains of Clostridium botulinum act by blocking the release of acetylcholine from peripheral nerve junctions. This specific action of the botulinum neurotoxins is now being exploited therapeutically to treat a variety of conditions involving involuntary muscle spasms. We aimed to develop a sensitive and specific enzyme-linked immunosorbent assay (ELISA) which may be used in parallel with the currently accepted mouse bioassay test for the determination of botulinum neurotoxin type A in therapeutic preparations. High titre polyclonal antitoxins and their biotin derivatives, highly labelled horseradish peroxidase (HRP)-antibody conjugates, and streptavidin-biotin-HRP complexes were prepared and used in a sandwich ELISA for the detection of pure neurotoxin and neurotoxin in therapeutic material. The ELISA utilized either a monoclonal or polyclonal antibody as capture agent and HRP-labelled IgG or F(ab')2 fragment as second antibody. The limit of detection was 4-8 pg of purified toxin/ml (gcv < 13%), equivalent to 1-2 mouse bioassay units/ml. The assay was used to evaluate therapeutic preparations and the results compared with the mouse bioassay. The lower limit of detection for a therapeutic preparation of BoTxA was 2-5 mouse bioassay units/ml. Although across different manufacturers and bulk products there was no correlation between immunologically detected neurotoxin and its biological activity in different therapeutic preparations (r = -0.44, p = 0.34, n = 8), the assay could be used to quantify neurotoxin in therapeutic preparations derived from the same bulk concentrate and manufacturer. The assay is relatively simple, and may be readily adapted to routine monitoring of BoTxA content in therapeutic preparations.

Technical influences on immunophenotyping by flow cytometry. The effect of time and temperature of storage on the viability of lymphocyte subsets.

The typing of lymphocyte subsets may be influenced by a variety of technical influences including the duration and temperature of sample storage and the method used for staining samples. We have extended a previous study examining the effect of storage conditions on the baseline values of a number of lymphocyte subsets. EDTA-anticoagulated samples from 13 HIV-1-positive and 15 healthy laboratory controls were analyzed for a number of lymphocyte subsets (CD3+, CD4+/CD3+, and CD8+/CD3+ T cells and CD19+ B cells) (whole blood lysis method, Becton-Dickinson FACScan flow cytometer and reagents) at 0, 24, 48, 72, and 96 h after storage at 4 degrees C, 17 degrees C or 21 degrees C. During storage at both 4 degrees C and 21 degrees C, there were significant changes in baseline values of the majority of lymphocyte subsets and some of these were related to the HIV status of the donor. The optimum temperature for storage in our system appeared to be around 17 degrees C in both our study groups. We have also used propidium iodide in order to discriminate between viable and non-viable cells during flow cytometry of lymphocytes from eight HIV-1-positive and five control subjects. The results show that for both HIV-positive and control samples stored at 4 degrees C, and for control subjects at 21 degrees C, the changes in baseline values of lymphocyte subsets observed were not due to selective loss of particular subsets arising from cell death during storage. However, there was substantial loss of cells from all three subsets in HIV-positive subjects during storage at 21 degrees C, with loss of CD8+ and CD3+ T cells being more significant than loss of CD4+ T cells.

Double-staining artefact observed in certain individuals during dual-colour immunophenotyping of lymphocytes by flow cytometry.

An unusual double-staining artefact has been repeatedly observed in certain individuals (both HIV-positive and healthy laboratory controls) during 2-colour immunophenotyping of lymphocytes by flow cytometry after red cell lysis. This artefact can falsely suggest co-expression of CD4 and CD8, as well as some of the other monoclonal antibody pairs commonly used in the typing of lymphocytes. It is caused by a serum factor associated with the ammonium-sulphate-precipitated and gamma-globulin fractions. It appears to be fairly common in the population (being found in 6 out of 105 of our healthy laboratory controls and in 42 out of 247 of our HIV-1 seropositive subjects); it may be genetically determined, but may be acquired in some. It is a potential source of error in lymphocyte immunophenotyping, which can be avoided by simple adjustments to the recommended techniques.

The effect of the temperature and duration of sample storage on the measurement of lymphocyte subpopulations from HIV-1-positive and control subjects.

EDTA-anticoagulated blood samples from 19 HIV-1-positive subjects and 13 healthy laboratory worker controls were analysed for three lymphocyte subpopulations (CD3, CD4, CD8 T cells) (lysed whole blood method, Becton Dickinson FACScan flow cytometer) at 0, 24, 48, 72 and 96 h after venesection, having been stored at either 4 degrees C, 12 degrees C, 16 degrees C or 21 degrees C. In samples stored at 4 degrees C and 12 degrees C there was a significant fall in both %CD3 and %CD 4, and a significant rise in %CD8. At 16 degrees C the %CD8 remained stable, while there were marginal rises in %CD3 and %CD4. At 21 degrees C, the %CD8 again remained stable, while %CD3 and %CD4 rose significantly with time. These trends were independent of HIV-1 status. At each temperature studied, the rates of change of lymphocyte subpopulations were independent of each other. These results suggest that a temperature range of 14-16 degrees C may be optimal for sample storage prior to measurement of T cell subsets. They emphasise the importance of strict control on conditions if samples are to be kept for any length of time before analysis.

Low affinity antibodies against collagen type II are associated with pathology in collagen-induced arthritis in mice.

The relationship between the functional affinity of antibodies against type II collagen (CII) and the development of arthritis was studied in mice with collagen-induced arthritis. The responses of DBA/1 strain mice were compared with those of mice selectively bred to produce antibodies of high functional affinity (HA mice) and low functional affinity (LA mice). HA and LA mice did not develop arthritis in response to immunization with CII whereas 86% of DBA/1 mice did, with 33% showing severe and 53% mild disease. Anti-CII antibodies of the highest titre, the lowest functional affinity, and the greatest affinity heterogeneity were associated with the development of the severest arthritis in DBA/1 mice: even in DBA/1 mice with moderate or no disease the amount of antibody and heterogeneity were higher and functional affinity lower than in either HA or LA mice. Antibodies of the G1, 2a, 2b and 3 subclasses were produced in all mice, and none of these alone accounted for the overall difference in IgG antibody titres or affinity in the groups of mice. Antibodies of the IgG2a subclass showed the closest association with the development of arthritis in the different groups. It is concluded that anti-CII antibodies of low functional affinity, and presumably also of the IgG2a subclass, influence the disease process in collagen arthritis.