Comparison of modeling methods to determine liver-to-blood inocula and parasite multiplication rates during controlled human malaria infection.

Controlled human malaria infection is used to measure efficacy of candidate malaria vaccines before field studies are undertaken. Mathematical modeling using data from quantitative polymerase chain reaction (qPCR) parasitemia monitoring can discriminate between vaccine effects on the parasite's liver and blood stages. Uncertainty regarding the most appropriate modeling method hinders interpretation of such trials. We used qPCR data from 267 Plasmodium falciparum infections to compare linear, sine-wave, and normal-cumulative-density-function models. We find that the parameters estimated by these models are closely correlated, and their predictive accuracy for omitted data points was similar. We propose that future studies include the linear model.

Morphometric evaluation of duodenal biopsies in celiac disease.

OBJECTIVES: The Marsh classification is a semiquantitative method for the diagnosis and monitoring of changes in duodenal biopsies in celiac disease. We have explored the possibility that quantitative changes in villous area and crypt length (morphometry) may provide better information on changes in duodenal morphology, particularly after the introduction of a gluten-free diet. METHODS: We measured villous height, apical and basal villous widths, and crypt length in 57 adults with celiac disease and 83 control subjects. Villous area was calculated as a trapezoid approximation. Serial changes in villous area and crypt length were determined at regular intervals for up to 4 years after the introduction of a gluten-free diet. Morphometric changes were also correlated with Marsh grade, self-reported adherence to a gluten-free diet, and changes in celiac serology. RESULTS: The gluten-free diet resulted in a progressive increase in villous area and a progressive decrease in crypt length. Morphometric improvement reached a plateau after 6-12 months with mean villous area attaining a value approximately half that of control subjects. Morphometric data were more sensitive than Marsh grade. Improvement in morphometric indices was significantly associated with the disappearance of anti-endomysial IgA antibody but not with dietary compliance. CONCLUSIONS: Morphometry is a sensitive way to document changes in duodenal biopsies in celiac disease. In adults treated with a gluten-free diet, it is uncommon for villous area to return to values observed in control subjects, but morphometric improvement is associated with the disappearance of anti-endomysial IgA antibody.

Multiple functions of human T cells generated by experimental malaria challenge.

Protective immunity generated following malaria infection may be comprised of Ab or T cells against malaria Ag of different stages; however, the short-lived immunity that is observed suggests deficiency in immune memory or regulatory activity. In this study, cellular immune responses were investigated in individuals receiving Plasmodium falciparum sporozoite challenge by the natural (mosquito bite) route as part of a malaria vaccine efficacy trial. Parasitemia, monitored by blood film microscopy and PCR, was subsequently cleared with drugs. All individuals demonstrated stable IFN-gamma, IL-2 and IL-4 ex vivo ELISPOT effector responses against P. falciparum-infected RBC (iRBC) Ag, 28 and 90 days after challenge. However, infected RBC-specific central memory responses, as measured by IFN-gamma cultured ELISPOT, were low and unstable over time, despite CD4(+) T cells being highly proliferative by CFSE dilution, and showed an inverse relationship to parasite density. In support of the observation of poor memory, co-culture experiments showed reduced responses to common recall Ag, indicating malaria-specific regulatory activity. This activity could not be accounted for by the expression of IL-10, TGF-beta, FOXP3 or CTLA-4, but proliferating T cells expressed high levels of CD95, indicating a pro-apoptotic phenotype. Lastly, there was an inverse relationship between FOXP3 expression, when measured 10 days after challenge, and ex vivo IFN-gamma measured more than 100 days later. This study shows that malaria infection elicits specific Th1 and Th2 effector cells, but concomitant weak central memory and regulatory activity, which may help to explain the short-lived immunity observed.

Liver disease in the young adult: the challenges and rewards.

Increasing numbers of children are surviving into adulthood with a diagnosis of liver disease or having undergone liver transplantation. This population presents some challenges for the adult hepatologist, and a formal transition service clearly improves outcomes for patients in this group. Evidence of ongoing neurological development in young people up to the age of 25 years exists, and understanding these physiological processes is important in overcoming some of the challenges that caring for this population presents. A well designed transition service is key to maximising potential for these patients, and should enable young people to take control of their illness and achieve their life goals.

Crypt fission peaks early during infancy and crypt hyperplasia broadly peaks during infancy and childhood in the small intestine of humans.

BACKGROUND: Postnatal growth of the small intestine occurs by crypt hyperplasia and by the less recognised mechanism of crypt fission. How the small intestine grows is largely extrapolated from animals and is poorly described in humans. AIM: To investigate crypt fission and crypt hyperplasia as mechanisms of intestinal growth in humans. PATIENTS AND METHODS: Proximal intestinal samples were taken from 3 neonates at surgical anastomosis, and duodenal biopsies were taken at endoscopy from 16 infants (mean age 0.7, range 0.3-1.7 years), 14 children (mean age 7.9, range 2.4-16.2 years), and 39 adults. Morphometric measures of villous area, crypt length (measure of crypt hyperplasia), and percentage of bifid crypts (measure of crypt fission) were assessed by a microdissection technique. RESULTS: Mean crypt fission rates in neonates, infants, children, and adults were 7.8%, 15%, 4.9%, and 1.7%, respectively. In particular, crypt fission peaked at 18% in 5 infants from 6 to 12 months of age. Mean crypt length was 123 microm in neonates, 287 microm in infants, 277 microm in children, and 209 microm in adults. Thus, crypt hyperplasia had a broad peak during infancy and childhood. CONCLUSIONS: We conclude that crypt fission was present predominantly during infancy, and crypt hyperplasia occurred during both infancy and childhood.

MAIT cells are activated during human viral infections.

Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation-driving cytokine release and Granzyme B upregulation-is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/ß. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology.

The effect of keratinocyte growth factor on tumour growth and small intestinal mucositis after chemotherapy in the rat with breast cancer.

PURPOSE: Mucositis from cancer chemotherapy is a common problem for which there is no definitive treatment. It produces significant morbidity and occasional mortality. Prevention and successful treatment could significantly enhance the quality of life of patients, and improve survival; however any potential preventative agent must not enhance tumour growth. The aims of this study were to assess the effect of keratinocyte growth factor (KGF) on breast tumour growth, and in preventing small intestinal mucositis induced by methotrexate (MTX). METHODS: Tumour-bearing rats received KGF or saline for 5 days prior to either MTX or saline treatment, and were killed 24 h after the last MTX injection. The weights of the tumour, small and large intestines, and liver were recorded. Apoptosis was assessed by TUNEL assay in the tumour and jejunum. Intestinal morphometry was used to assess villus area, crypt length and mitotic crypt count. Tumour proliferation was assessed by mitotic count. RESULTS: KGF increased the weight of the small intestine prior to chemotherapy but the weight was not maintained after chemotherapy. KGF synergized with MTX to increase apoptosis in both intestinal crypts and the breast cancer. KGF also reduced tumour size. CONCLUSIONS: We conclude that KGF had a modest effect on intestinal growth prior to chemotherapy. It did not protect the gut from mucositis, nor did it worsen morphometry. It reduced tumour size.

A human Phase I/IIa malaria challenge trial of a polyprotein malaria vaccine.

We examined the safety, immunogenicity and efficacy of a prime-boost vaccination regime involving two poxvirus malaria subunit vaccines, FP9-PP and MVA-PP, expressing the same polyprotein consisting of six pre-erythrocytic antigens from Plasmodium falciparum. Following safety assessment of single doses, 15 volunteers received a heterologous prime-boost vaccination regime and underwent malaria sporozoite challenge. The vaccines were safe but interferon-γ ELISPOT responses were low compared to other poxvirus vectors, despite targeting multiple antigens. There was no vaccine efficacy as measured by delay in time to parasitaemia. A number of possible explanations are discussed, including the very large insert size of the polyprotein transgene.

Impact on malaria parasite multiplication rates in infected volunteers of the protein-in-adjuvant vaccine AMA1-C1/Alhydrogel+CPG 7909.

BACKGROUND: Inhibition of parasite growth is a major objective of blood-stage malaria vaccines. The in vitro assay of parasite growth inhibitory activity (GIA) is widely used as a surrogate marker for malaria vaccine efficacy in the down-selection of candidate blood-stage vaccines. Here we report the first study to examine the relationship between in vivo Plasmodium falciparum growth rates and in vitro GIA in humans experimentally infected with blood-stage malaria. METHODS: In this phase I/IIa open-label clinical trial five healthy malaria-naive volunteers were immunised with AMA1/C1-Alhydrogel+CPG 7909, and together with three unvaccinated controls were challenged by intravenous inoculation of P. falciparum infected erythrocytes. RESULTS: A significant correlation was observed between parasite multiplication rate in 48 hours (PMR) and both vaccine-induced growth-inhibitory activity (Pearson r = -0.93 [95% CI: -1.0, -0.27] P = 0.02) and AMA1 antibody titres in the vaccine group (Pearson r = -0.93 [95% CI: -0.99, -0.25] P = 0.02). However immunisation failed to reduce overall mean PMR in the vaccine group in comparison to the controls (vaccinee 16 fold [95% CI: 12, 22], control 17 fold [CI: 0, 65] P = 0.70). Therefore no impact on pre-patent period was observed (vaccine group median 8.5 days [range 7.5-9], control group median 9 days [range 7-9]). CONCLUSIONS: Despite the first observation in human experimental malaria infection of a significant association between vaccine-induced in vitro growth inhibitory activity and in vivo parasite multiplication rate, this did not translate into any observable clinically relevant vaccine effect in this small group of volunteers. TRIAL REGISTRATION: ClinicalTrials.gov [NCT00984763].

Deficiency of 6B11+ invariant NK T-cells in celiac disease.

Immunoregulatory NK T-cells are deficient in certain autoimmune diseases. The purpose of this study was to investigate any deficiency of immunoregulatory NK T-cells in celiac disease. NK T-cells were identified by flow cytometry with 6B11 and V alpha 24 markers in blood from 18 normal and 12 celiac subjects. Blood mononuclear cells were stimulated with anti-CD3/CD28 antibodies and intracellular cytokines assessed at 4 h in seven normal and eight celiac subjects. V alpha 24/GAPDH mRNA was quantitated in duodenal biopsies by real time PCR in 17 control and 13 celiac subjects. NK T-cells in celiac subjects were reduced to 30% of those in normal subjects. Intracellular IL-4, IL-10 and IL-13 increased significantly by 33-41% in normal subjects, but did not change in celiac subjects. V alpha 24/GAPDH mRNA from celiac subjects was reduced to 5% of levels in control subjects. We conclude that immunoregulatory NK T-cells are deficient in celiac disease.

Evidence of blood stage efficacy with a virosomal malaria vaccine in a phase IIa clinical trial.

BACKGROUND: Previous research indicates that a combination vaccine targeting different stages of the malaria life cycle is likely to provide the most effective malaria vaccine. This trial was the first to combine two existing vaccination strategies to produce a vaccine that induces immune responses to both the pre-erythrocytic and blood stages of the P. falciparum life cycle. METHODS: This was a Phase I/IIa study of a new combination malaria vaccine FFM ME-TRAP+PEV3A. PEV3A includes peptides from both the pre-erythrocytic circumsporozoite protein and the blood-stage antigen AMA-1. This study was conducted at the Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, Oxford, UK. The participants were healthy, malaria naïve volunteers, from Oxford. The interventions were vaccination with PEV3A alone, or PEV3A+FFM ME-TRAP. The main outcome measure was protection from malaria in a sporozoite challenge model. Other outcomes included measures of parasite specific immune responses induced by either vaccine; and safety, assessed by collection of adverse event data. RESULTS: We observed evidence of blood stage immunity in PEV3A vaccinated volunteers, but no volunteers were completely protected from malaria. PEV3A induced high antibody titres, and antibodies bound parasites in immunofluorescence assays. Moreover, we observed boosting of the vaccine-induced immune response by sporozoite challenge. Immune responses induced by FFM ME-TRAP were unexpectedly low. The vaccines were safe, with comparable side effect profiles to previous trials. Although there was no sterile protection two major observations support an effect of the vaccine-induced response on blood stage parasites: (i) Lower rates of parasite growth were observed in volunteers vaccinated with PEV3A compared to unvaccinated controls (p = 0.012), and this was reflected in the PCR results from PEV3A vaccinated volunteers. These showed early control of parasitaemia by some volunteers in this group. One volunteer, who received PEV3A alone, was diagnosed very late, on day 20 compared to an average of 11.8 days in unvaccinated controls. (ii). Morphologically abnormal parasites were present in the blood of all (n = 24) PEV3A vaccinated volunteers, and in only 2/6 controls (p = 0.001). We describe evidence of vaccine-induced blood stage efficacy for the first time in a sporozoite challenge study.

Activation of beta-catenin in the stem cell region of crypts during growth of the small intestine in infant rats.

The Wnt/beta-catenin pathway has been proposed as promoting intestinal stem cell division. Wnt ligands activate cytoplasmic beta-catenin and increase nuclear translocation of beta-catenin that binds to the Tcf-4 transcription factor. The aim of this study was to investigate beta-catenin expression in the stem cell region of crypts during intestinal growth in rats. Litters of DAxPVG/c rats were humanely killed at 7, 14, 21, 35, and 72 days of life. beta-Catenin and Tcf-4 were quantified by immunoperoxidase staining and image analysis with cumulative signal analysis. Cytoplasmic and nuclear expression of beta-catenin peaked nearly 2-fold at day 14 (versus day 7) of life in the stem cell region of intestinal crypts. Tcf-4 nuclear expression peaked earlier at 7 days and was lower thereafter with age. We conclude that the Wnt/beta-catenin pathway is activated in the stem cell region of intestinal crypts during growth of the small intestine.

Deficiency of invariant NK T cells in Crohn's disease and ulcerative colitis.

The aim of this study was to investigate whether immunoregulatory invariant NK T cells are deficient in Crohn's disease or ulcerative colitis. Blood was collected for flow cytometry from 106 Crohn's disease, 91 ulcerative colitis, and 155 control subjects. Invariant NK T cells were assessed by Valpha24 and (alpha-galactosylceramide/CD1d tetramer markers. Intracellular cytokine was measured after in vitro anti-CD3 antibody stimulation. Valpha24+ T cells were quantified in ileocolonic biopsies as mRNA by real-time PCR and by immunofluorescence. Circulating invariant NK T cells were 5.3% of the control levels in Crohn's (P < 0.001) and 7.9% of the control levels in ulcerative colitis (P < 0.001). Interleukin-4 production was impaired in Crohn's disease and ulcerative colitis. Intestinal Valpha24 mRNA expression was 7% in Crohn's disease (P < 0.05) and 9% in ulcerative colitis (P < 0.05). Intestinal Valpha24+ T cells were 23% in Crohn's disease but not reduced in ulcerative colitis. We conclude that invariant NK T cells are deficient in Crohn's disease and in ulcerative colitis.

Postnatal epithelial growth of the small intestine in the rat occurs by both crypt fission and crypt hyperplasia.

Studies of growth of the small intestine have largely concentrated on crypt hyperplasia rather than crypt fission. The aim of this study was to investigate quantitatively both crypt fission and crypt hyperplasia. DAxPVG/c rats were killed at 7, 11, 14, 17, 19, 21, 25, 55, and 72-73 days of life. Samples of jejunum at one third of the intestinal length were taken for morphometry (villous area, crypt area, percentage of bifid crypts, and crypt mitotic count) by microdissection. Growth factors and their receptors were assessed by oligonucleotide microarray. Crypt fission was 10.5%, 5.2%, and 1.5% at days 11, 25, and 72-73 of life, respectively. Crypt hyperplasia increased from day 21. No conventional growth factor was identified during crypt fission. We conclude that crypt fission contributes to growth of the small intestine prior to weaning and crypt hyperplasia to growth after weaning.

Safety, immunogenicity, and efficacy of prime-boost immunization with recombinant poxvirus FP9 and modified vaccinia virus Ankara encoding the full-length Plasmodium falciparum circumsporozoite protein.

Heterologous prime-boost immunization with DNA and various recombinant poxviruses encoding malaria antigens is capable of inducing strong cell-mediated immune responses and partial protection in human sporozoite challenges. Here we report a series of trials assessing recombinant fowlpox virus and modified vaccinia virus Ankara encoding the Plasmodium falciparum circumsporozoite protein in various prime-boost combinations, doses, and application routes. For the first time, these vaccines were administered intramuscularly and at doses of up to 5 x 10(8) PFU. Vaccines containing this antigen proved safe and induced modest immune responses but showed no evidence of efficacy in a sporozoite challenge.

Effect of interleukin-11 on ameliorating intestinal damage after methotrexate treatment of breast cancer in rats.

Gastrointestinal mucositis after cancer chemotherapy is an increasing problem that is essentially untreatable once established, although it gradually remits. The aim of this study was to investigate the time-course and effect of interleukin-11 (IL-11) on apoptosis and intestinal morphometry as measures of mucositis. Female DA rats were implanted subcutaneously with syngeneic breast cancer and treated with methotrexate (MTX). Intestinal morphometry was used to assess villus area, crypt length, and mitotic count per crypt. Apoptosis was assessed by TUNEL assay in the tumor and jejunum. Tumor proliferation was assessed by mitotic count. The time-course study showed that MTX increased apoptosis by 28-fold in the crypts of the small intestine and by 3-fold in the tumor, and peaked at 6 hr after chemotherapy. IL-11 (100 microg/kg/twice daily subcutaneously) maintained intestinal weight, and reduced the severity of mucositis, as measured by villus area, crypt length, and mitotic count per crypt. IL-11 at higher doses (200 microg and 400 microg/kg/twice daily subcutaneously), did not further improve villus area, crypt length, and mitotic count per crypt. IL-11 did not affect tumor apoptosis or proliferation. We conclude that IL-11 attenuated mucositis by maintaining intestinal weight and morphometry. IL-11 did not prevent apoptosis, but rather induced compensatory crypt cell proliferation.