An introduction to clinical trial design.

Clinicians and other decision makers in healthcare use results from clinical trials to inform practice. Interpretation of clinical trial results can be challenging, as weaknesses in trial design, data collection, analysis or reporting, can compromise the usefulness of results. A good working knowledge of clinical trial design is essential to expertly interpret and determine the validity and generalizability of the results. This manuscript will give a brief overview of clinical trial design including the strengths and limitations of various approaches. The focus will be on confirmatory clinical trials.

Effects of maternal dietary egg intake during early lactation on human milk ovalbumin concentration: a randomized controlled trial.

BACKGROUND: There is limited understanding of how maternal diet affects breastmilk food allergen concentrations, and whether exposure to allergens through this route influences the development of infant oral tolerance or sensitization. OBJECTIVE: To investigate how maternal dietary egg ingestion during early lactation influences egg protein (ovalbumin) levels detected in human breastmilk. METHODS: In a randomized controlled trial, women were allocated to a dietary group for the first six weeks of lactation: high-egg diet (> 4 eggs per week), low-egg diet (one-three eggs per week) or an egg-free diet. Breastmilk samples were collected at 2, 4 and 6 weeks of lactation for the measurement of ovalbumin. The permeability of the mammary epithelium was assessed by measuring the breastmilk sodium : potassium ratio. Egg-specific IgE and IgG4 were measured in infant plasma at 6 weeks, and prior to the introduction of egg in solids at 16 weeks. RESULTS: Average maternal egg ingestion was associated with breastmilk ovalbumin concentration. Specifically, for each additional egg ingested per week, there was an average 25% increase in ovalbumin concentration (95% CI: 5-48%, P = 0.01). Breastmilk ovalbumin concentrations were significantly higher in the 'high-egg' group (> 4 eggs per week) compared with the 'egg-free' group (P = 0.04). However, one-third of women had no breastmilk ovalbumin detected. No detectable associations were found between mammary epithelium permeability and breastmilk ovalbumin concentrations. Infant plasma egg-specific IgG4 levels were also positively associated with maternal egg ingestion, with an average 22% (95% CI: 3-45%) increase in infant egg-specific IgG4 levels per additional egg consumed per week (P = 0.02). CONCLUSIONS AND CLINICAL RELEVANCE: Increased maternal egg ingestion is associated with increased breastmilk ovalbumin, and markers of immune tolerance in infants. These results highlight the potential for maternal diet to benefit infant oral tolerance development during lactation.

The Platform Trial In COVID-19 Priming and BOOsting (PICOBOO): the immunogenicity, reactogenicity, and safety of different COVID-19 vaccinations administered as a second booster (fourth dose) in AZD1222 primed individuals aged 50-<70 years old.

OBJECTIVES: PICOBOO is a randomised, adaptive trial evaluating the immunogenicity, reactogenicity, and safety of COVID-19 booster strategies. We report data for second boosters among individuals 50-<70 years old primed with AZD1222 (50-<70y-AZD1222) until Day 84. METHODS: Contributed equally as first authors.Immunocompetent adults who received any first booster >three months prior were eligible. Participants were randomly allocated to BNT162b2, mRNA-1273 or NVX-CoV2373 1:1:1. The concentrations of ancestral anti-spike immunoglobulin was summarised as the geometric mean concentrations (GMC). Reactogenicity and safety outcomes were captured. Additional analyses including neutralising antibodies were performed on a subset. ACTRN12622000238774. RESULTS: Between Mar 2022-Aug 2023, 743 participants were recruited and had D28 samples; 155 belonged to the 50-<70y-AZD1222 stratum. The mean adjusted GMCs (95% credible intervals) were 20,690 (17,555-23,883), 23,867 (20,144-27,604) and 8,654 (7,267-9,962) U/mL at D28 following boosting with BNT162b2, mRNA-1273 and NVX-CoV2372, respectively, and 10,976 (8,826-13,196), 15,779 (12,512-19,070) and 6,559 (5 220-7 937) U/mL by D84. IgG against Omicron BA.5 was 2.7-2.9 times lower than the ancestral strain. Limited neutralisation against Omicron subvariants was found following all vaccines. Severe reactogenicity events were <4%. CONCLUSIONS: All vaccines were immunogenic with more rapid waning after mRNA vaccines. These data support boosting with vaccines with greater specificity for circulating Omicron subvariants.

A cyclophilin function in Hsp90-dependent signal transduction.

Cpr6 and Cpr7, the Saccharomyces cerevisiae homologs of cyclophilin-40 (CyP-40), were shown to form complexes with Hsp90, a protein chaperone that functions in several signal transduction pathways. Deletion of CPR7 caused severe growth defects when combined with mutations that decrease the amount of Hsp90 or Sti1, another component of the Hsp90 chaperone machinery. The activities of two heterologous Hsp90-dependent signal transducers expressed in yeast, glucocorticoid receptor and pp60(v-src) kinase, were adversely affected by cpr7 null mutations. These results suggest that CyP-40 cyclophilins play a general role in Hsp90-dependent signal transduction pathways under normal growth conditions.

Cns1 is an essential protein associated with the hsp90 chaperone complex in Saccharomyces cerevisiae that can restore cyclophilin 40-dependent functions in cpr7Delta cells.

Saccharomyces cerevisiae harbors two cyclophilin 40-type enzymes, Cpr6 and Cpr7, which are components of the Hsp90 molecular chaperone machinery. Cpr7 is required for normal growth and is required for maximal activity of heterologous Hsp90-dependent substrates, including glucocorticoid receptor (GR) and the oncogenic tyrosine kinase pp60(v-src). In addition, it has recently been shown that Cpr7 plays a major role in negative regulation of the S. cerevisiae heat shock transcription factor (HSF). To better understand functions associated with Cpr7, a search was undertaken for multicopy suppressors of the cpr7Delta slow-growth phenotype. The screen identified a single gene, designated CNS1 (for cyclophilin seven suppressor), capable of suppressing the cpr7Delta growth defect. Overexpression of CNS1 in cpr7Delta cells also largely restored GR activity and negative regulation of HSF. In vitro protein retention experiments in which Hsp90 heterocomplexes were precipitated resulted in coprecipitation of Cns1. Interaction between Cns1 and the carboxy terminus of Hsp90 was also shown by two-hybrid analysis. The functional consequences of CNS1 overexpression and its physical association with the Hsp90 machinery indicate that Cns1 is a previously unidentified component of molecular chaperone complexes. Thus far, Cns1 is the only tetratricopeptide repeat-containing component of Hsp90 heterocomplexes found to be essential for cell viability under all conditions tested.

Parental pre-pregnancy BMI is a dominant early-life risk factor influencing BMI of offspring in adulthood.

OBJECTIVE: We examined parental and early-life variables in order to identify risk factors for adulthood overweight and obesity in offspring. We report here on the longitudinal prevalence of overweight and obesity in Australian children born between 1989 and 1991 and followed from birth to age 22. METHODS: Data were analysed on 1355 participants from the Western Australian Pregnancy Cohort (Raine) Study, with anthropometry collected during pregnancy, at birth, one year and at three yearly intervals thereafter. Multivariate analyses and cross-sectional logistic regression quantified the timing and contribution of early-life risk factors for overweight and obesity in young-adulthood. RESULTS: At five years of age 12.6% of children were overweight and 5.2% were obese. By early adulthood, the prevalence of obesity had increased to 12.8%, whilst overweight remained relatively stable at 14.2% (range from early childhood to adulthood 11-16%). Parental pre-pregnancy body mass index (BMI) was the strongest determinant of adult offspring BMI. Although rapid first year weight gain was associated with increased offspring BMI, the impact of first year weight-gain diminished over childhood, whilst the impact of parental BMI increased over time. CONCLUSIONS: Parental pre-pregnancy BMI and rapid early-life weight gain predispose offspring to obesity in adulthood.

Nucleotide sequence of the chicken HMGI-C cDNA and expression of the HMGI-C and IGF1 genes in autosomal dwarf chicken embryos.

Mutations in the genes for high mobility group protein I-C (HMGI-C) and insulin-like growth factor 1 (IGF1) are known to be responsible for dwarf phenotypes in the mouse. Because the locus for autosomal dwarfism (adw) in the chicken maps to a region which is syntenic to a region in the human and mouse in which the HMGI-C and IGF1 genes are located, HMGI-C and IGF1 are likely candidate genes for adw in the chicken. In this study their possible role in the establishment of this phenotype has been investigated. We have cloned and sequenced the complete coding region of the chicken HMGI-C cDNA. Comparison with its human counterpart revealed a nucleotide sequence conservation of 84%. Only nine amino acids are present principally in the N-terminal segment before the first DNA-binding domain. Northern blot analysis showed no difference in the expression of the HMGI-C gene between adw and wild-type chicken embryos. Also no mutations in either the HMGI-C or the IGF1 RNA nucleotide sequence were detected in adw chicken embryos.

Purification of recombinant adeno-associated virus vectors by column chromatography and its performance in vivo.

Recombinant adeno-associated virus (AAV) holds much promise for human gene therapy. While evidence indicates that AAV mediates long-term gene transfer in several different tissues, difficulty in preparing and purifying this viral vector in large quantities remains a major obstacle for evaluating AAV vectors in clinical trials. The current method of purification, based on sedimentation through cesium chloride, is not scaleable and yields product of insufficient quality. In this article we report a new technique for purifying AAV, using a fully closed two-column chromatography system. Yields of AAV vectors purified by this method are high, potency is increased, and the purity of column-purified preparations is substantially improved. We previously reported a novel method to generate AAV based on an AAV Rep/Cap-containing cell line (B50) and an Ad-AAV hybrid virus, which is amenable to scale-up in bioreactors. By combining the new, fully scaleable purification process we report here with the B50/hybrid production method, it would be feasible to prepare AAV vectors to the scale and purity required for clinical and potential commercial applications.

Assessment of antibody production in sex-linked and autosomal dwarf chickens.

The humoral immune response was examined in two dwarf and one control strain of Single Comb White Leghorn chicks. Circulating antibody levels in response to immunization with varying concentrations of sheep erythrocytes were determined following primary immunization and again following a secondary antigen challenge. The males of the sex-linked dwarf strain produced significantly less antibody to lower antigen dosages in both the primary and secondary response but as antigen dose was increased the humoral response was no longer depressed below that of the controls. The antibody response of females carrying the dw gene was not significantly impaired over the entire antigen dose range nor was that of either sex of the autosomal dwarf strain. Similar results were obtained when the numbers of splenic plaque-forming cells (PFC's) were examined. However, in this assay both the males and females of the sex-linked dwarf strain demonstrated significantly impaired plaque-forming activity. No differences were found between the control and autosomal dwarf strains in their ability to generate PFC's.

The effect of synthetic thymulin on cell surface marker expression by avian T-cell precursors.

The effects of the in vitro exposure of avian bone marrow (BM) cells and thymocytes to synthetic thymulin were studied. Two T-cell differentiation markers, PNA binding site and CT-1a expression, were used to examine cell maturation. Enhanced PNA binding to both BM cells and thymocytes resulted following an in vitro thymulin exposure but cell proliferation was not affected. Scatchard analysis supported the conclusion that PNA binding affinity was significantly increased and thus responsible for the observed increase in PNA binding. Flow cytometric analysis suggested that the induced PNA+ thymocyte population may be a different population from the one exhibiting enhanced CT-1a expression following thymulin exposure. Taken together, the observations suggest that cells can express their further differentiation states without undergoing proliferation following in vitro thymulin stimulation.

Evidence for the direct action of thymulin on avian NK cells.

The ability of thymulin to directly enhance NK cell-mediated cytotoxicity was examined. Specific cell population depletions were done in K and SLD chicken splenocyte preparations using anti-CD3, CD4, and CD8 monoclonal antibodies and secondary complement-fixing polyclonal antibodies. The remaining cells were incubated overnight with in vitro treatments of thymulin and IFN-gamma, either separately or together, followed by an assay for cytotoxicity. Although the control K-strain had higher overall NK cell-mediated cytotoxicity than the thymulin-deficient SLD-strain, the following trends were seen in both strains. Thymulin continued to enhance NK activity following CD4 or CD3 cell depletion, but not after CD8 or CD8 and CD4 cell depletion. Since avian NK cells express CD8 alpha, but not CD3 or CD4 on their surface, these results suggest that the ability of in vitro thymulin treatments to enhance NK activity is not mediated by T-cells but may be due to direct effects on NK cells.

Differential effects of thyroxine on immune development and autoimmune thyroiditis in the obese strain chicken.

The effects of dietary thyroxine (T4) supplementation for specific periods on the early development of the primary lymphoid organs and spontaneous autoimmune thyroiditis (SAT) was examined in the Obese (OS) strain of chicken. Effects of the treatments on concentrations of serum growth hormone (GH) and testosterone were also determined. All treatment groups were examined at 6 weeks. T4 supplementation did not affect serum testosterone or GH concentrations. However T4 given for the first three weeks resulted in significantly increased bursa weights, no change in thymic weights, significantly decreased lymphoid infiltration of the thyroid and reduced thyroglobulin autoantibody levels (TgAAb). T4 supplementation for the full six weeks resulted in no change in bursal weight, significantly increased thymic weight, significantly decreased lymphoid infiltration of the thyroid, and reduced TgAAb. These results suggest that the effects of T4 supplementation on SAT and immune development are dependent on the interval during which it is administered and that testosterone and GH probably do not mediate these effects.

Triiodothyronine affects mitogen responsiveness in sex-linked dwarf and Cornell K strain chickens.

The response of lymphocytes to concanavalin A (Con A) was examined in the functionally hypothyroid SLD strain and the normal K strain chickens which were fed diets with or without T3 supplementation (0.1, 0.5 and 1.0 ppm) since the time of hatch. Peripheral blood lymphocytes (PBL) from 3 and 12 week old male and female chickens were incubated with Con A for 60 h. Mitogenic responsiveness was assessed by measuring the uptake of 3H-thymidine during the last 24 h of incubation. There was no difference in the mitogen response between male and female chickens. The mitogen responsiveness of PBL from the K strain tended to be greater than that from the SLD strain at both ages. The lowest dose of T3 (0.1 ppm) had no effect on the mitogen response of 3-week-old K strain chicks but caused an increase in mitogen response in 3-week-old SLD and in 12-week-old birds of both strains. Supplementation with 0.5 and 1.0 ppm T3 tended to decrease mitogen responsiveness in the K and SLD strain at both ages. The effect of treatment on thymus weights, bursa weights, and lymphocyte concentrations of the blood was also assessed.

Effect of dietary triiodothyronine on mixed-lymphocyte responsiveness in young male chickens.

The effect of dietary triiodothyronine (T3) supplementation (0, 0.1, 0.1, and 1.0 ppm of T3) on the mixed lymphocyte response (MLR) was examined in MHC-matched young Cornell K and sex-linked dwarf (SLD) strain cockerels at ages 6, 9, and 12 weeks. At all ages, MLR in SLD strain cockerels was significantly lower than in the K strain. Supplementation of 6-week-old cockerels with 0.01 and 0.1 ppm of T3 tended to decrease MLR in both strains, whereas the highest dose of T3 had the opposite effect in the K strain and no effect in the SLD strain compared to the control. No significant effects of T3 on MLR were observed in 9-week-old cockerels. A significant main treatment effect of T3 on MLR was observed in 12-week-old cockerels. At this age dietary T3 decreased MLR in both strains. These results show that allo-aggression, a functional parameter of cell-mediated immunity, can be affected by non-MHC controlled strain differences and by the iodothyronine state of these strains.

The enhancement of avian NK cell cytotoxicity by thymulin is not mediated by the regulation of IFN-gamma production.

Preincubation with either thymulin or IFN-gamma can enhance NK activity. In addition, overnight in vitro pre-treatment with thymulin and IFN-gamma increases NK activity further than either treatment alone. It has been hypothesized that thymulin increases the responsiveness of immune cells to IFN-gamma by either increasing the expression of IFN-gammaR or by increasing the production and/or secretion of IFN-gamma. The effects of thymulin on IFN-gamma production and secretion were examined in this study. While an overnight incubation with the polyclonal activator Con A increased the number of cells positive for intracellular IFN-gamma, a similar incubation with thymulin produced no change in the percentages of cells labeling positive for intracellular IFN-gamma when compared to the media control cells. In addition, IFN-gamma was not secreted by splenocytes following an overnight incubation with thymulin, but increased secretion was induced by Con A stimulation. Taken together, these results suggest that thymulin does not increase IFN-gamma production or induce IFN-gamma secretion by avian splenocytes.

Effect of triiodothyronine and in vitro growth hormone on avian interleukin-2.

One-day-old chickens were treated with varying levels of triiodothyronine (T3) added to the diet. At 28 days of age, the IL-2-like activity in the splenocyte culture supernatants were assessed. The lowest dose of T3 (0.1 ppm) enhanced IL-2-like activity while the highest dose (1.0 ppm) was significantly suppressive. The intermediate dose elicited varying effects. Recombinant chicken growth hormone (rcGH) was added to some cultures 24 h prior to IL-2 assay. In vitro rcGH significantly depressed the IL-2-like activity of splenocytes from animals given the low T3 diet. The addition of varying concentrations of T3 in vitro to splenocytes from non-T3-supplemented chickens had no effect on the IL-2-like activity. These results indicate that in vivo supplementation of low dietary T3 but not in vitro T3 is effective in enhancing avian IL-2-like activity. The addition of rcGH in vitro can modify this response.

Effect of growth hormone and thyroid hormone on autoimmune thyroiditis in obese chickens.

The effect of thyroxine (T4) and recombinant (rcGH) or purified pituitary-derived (pcGH) chicken growth hormone on the development of spontaneous autoimmune thyroiditis (SAT) was examined in the Obese strain (OS) chicken. Day-old OS chicks were randomly assigned to a control or 1.0 ppm T4 supplemented diet and a vehicle or 500 micrograms rcGH/kg BW daily injection, using a 2 x 2 factorial design. At 4 weeks, sera were analyzed for anti-thyroglobulin autoantibody (TgAAb) using a kinetics-based ELISA. Leucocytic infiltration of the thyroid was assessed using computer-based video imaging techniques. A close correlation between TgAAb and thyroid infiltration was seen with both being decreased (p < 0.05) by the T4/rcGH treatment. Neither the T4 or rcGH alone produced this effect and the rcGH treatment significantly elevated TgAAb. In a second experiment, all but the control group received 1.0 ppm T4 supplementation and two of the T4-treated groups received either 50 or 200 micrograms pcGH/kg BW by daily injection. As before, T4/pcGH significantly reduced TgAAb and thyroid infiltration. T4 alone produced no significant effects. These data support the conclusion that the combined treatment of T4 and cGH exert an immunomodulatory effect within a strain that is predisposed to autoimmune thyroiditis while GH treatment alone exacerbated the condition. These results also show that video imaging techniques can be used to evaluate the extent of histopathology present within the OS thyroid.

Graft-versus-host response in sex-linked dwarf, autosomal dwarf and control K strain chickens.

Two independent experiments were conducted to examine the ability of 1) the autosomal-dwarf (ADW) strain and 2) the sex-linked (SLD) strain chicken to make a Graft-versus-Host (GvH) response. In each experiment the GvH response of the dwarf strain was compared to the GvH response of a normal growing control strain, the Cornell K strain chicken. All 3 strains were homozygous B15/B15 at the major histocompatibility complex. GvH responsiveness of peripheral blood lymphocytes (PBL) from females of each strain was assessed at various intervals from 1.5 to 20 months of age using the chorio-allantoic membrane (CAM) assay. The GvH response in female chickens from the ADW strain was significantly higher than in K strain females after the birds had reached sexual maturity (after 5.5 months). The GvH response in females from the SLD strain was, however, significantly lower than in the K strain females throughout the experiment. All strains tended to have a biphasic GvH response. There was a significant increase in GvH responsiveness from 1.5 to 5.5 months of age in chicks from all strains. In the SLD and K strain chickens, this was followed by a drop in the GvH response until 8.5 months of age. In 16- to 20-month-old SLD and K strain hens, the ability to mount a GvH response returned to levels observed in younger (5.5 months) pullets. The GvH responsiveness of the ADW strain remained at a constant level from 5.5 to 12 months of age. However, a second peak in GvH responsiveness was observed in 16-month-old ADW strain hens. Strain differences in GvH responsiveness may be due to the known hormonal abnormalities in the dwarf strains. The suitability of these dwarf strains for the study of endocrine-immune interrelationships is discussed.

Effect of hypophysectomy and growth hormone on immune development in the domestic fowl.

The effect of hypophysectomy and recombinant growth hormone (rcGH) treatment on the growth and development of the immune system was investigated in young chickens. Flow cytometric analysis of cell surface markers revealed no changes in the proportion of thymocytes expressing CT-1a, CD4, and/or CD8 among any of the treatment groups. In contrast, the proportion of both single positive CD4 and CD8 peripheral blood lymphocytes (PBL) was altered in hypox birds treated with rcGH compared to the vehicle-treated group. Specifically, rcGH treatment produced a decrease in the proportion of CD8+ cells and an increase in the percentage of CD4+ PBL. There was little change in the labeling intensity of PBL or thymocytes associated with any treatment; however, double positive (CD4+CD8+) thymocytes from hypophysectomized chicks that were not given rcGH had increased fluorescence relative to rcGH supplemented hypox chicks. As expected, hypophysectomy reduced body, skeletal, and thymic growth. Treatment of hypox chicks with rcGH enhanced body weight while thymic weights were somewhat increased. Skeletal growth was not significantly altered by rcGH. Bursal growth appeared refractory to either treatment. These studies support the conclusion that growth hormone influences thymic growth and the maturation of thymus-derived lymphocytes. These results also demonstrate a biological activity for chicken growth hormone derived through recombinant technology.

Autoanti-thyroglobulin production in obese chickens: influence of age and sex as measured by ELISA.

The kinetics of serum autoantibody production against thyroglobulin (Tg) was examined in two strains of chicken using an enzyme-linked immunosorbant assay (ELISA). Both strains are homozygous for the B13 haplotype. The OS strain develops spontaneous autoimmune thyroiditis at several weeks of age while the Sp. C strain, which serves as the control for the OS strain, has virtually undetectable autoantibody levels as determined by hemagglutination assay (HA). Serum autoantibody levels in both strains were monitored bi-weekly from 6 through 14 weeks of age post-hatching, using both the ELISA and HA techniques. With the more sensitive ELISA, absolute serum autoantibody concentrations were determined and both the Sp. C and OS strains were found to have readily detectable serum autoantibodies against Tg; however, the OS did have significantly higher autoantibody levels than the Sp. C strain. In the latter strain, autoantibodies increased significantly with age while the pattern was somewhat reversed in the OS strain. The ELISA revealed that the decline of Tg autoantibodies with age in the OS strain was restricted primarily to males, with females maintaining constant levels of autoantibodies. In contrast, the HA detected only the differences in autoantibody levels between the OS and Sp. C strains.