Abstract
Alkaptonuria is an inherited disease caused by homogentisate 1,2-dioxygenase (HGD) deficiency. Circulating homogentisic acid (HGA) is elevated and deposits in connective tissues as ...ochronotic pigment. In this study, we aimed to define developmental and adult HGD tissue expression and determine the location and amount of gene activity required to lower circulating HGA and rescue the alkaptonuria phenotype.
We generated an alkaptonuria mouse model using a knockout-first design for the disruption of the HGD gene. Hgd tm1a −/− mice showed elevated HGA and ochronosis in adulthood. LacZ staining driven by the endogenous HGD promoter was localised to only liver parenchymal cells and kidney proximal tubules in adulthood, commencing at E12.5 and E15.5 respectively. Following removal of the gene trap cassette to obtain a normal mouse with a floxed 6th HGD exon, a double transgenic was then created with Mx1-Cre which conditionally deleted HGD in liver in a dose dependent manner. 20% of HGD mRNA remaining in liver did not rescue the disease, suggesting that we need more than 20% of liver HGD to correct the disease in gene therapy.
Kidney HGD activity which remained intact reduced urinary HGA, most likely by increased absorption, but did not reduce plasma HGA nor did it prevent ochronosis. In addition, downstream metabolites of exogenous 13C6-HGA, were detected in heterozygous plasma, revealing that hepatocytes take up and metabolise HGA.
This novel alkaptonuria mouse model demonstrated the importance of targeting liver for therapeutic intervention, supported by our observation that hepatocytes take up and metabolise HGA.
To determine whether adding bevacizumab, with or without mitomycin, to capecitabine monotherapy improves progression-free survival (PFS) in patients with metastatic colorectal cancer (mCRC) in an ...open-label, three-arm randomized trial.
Overall, 471 patients in Australia, New Zealand, and the United Kingdom with previously untreated, unresectable mCRC were randomly assigned to the following: capecitabine; capecitabine plus bevacizumab (CB); or capecitabine, bevacizumab, and mitomycin (CBM). We compared CB with capecitabine and CBM with capecitabine for progression-free survival (PFS). Secondary end points included overall survival (OS), toxicity, response rate (RR), and quality of life (QOL).
Median PFS was 5.7 months for capecitabine, 8.5 months for CB, and 8.4 months for CBM (capecitabine v CB: hazard ratio HR, 0.63; 95% CI, 0.50 to 0.79; P < .001; C v CBM: HR, 0.59; 95% CI, 0.47 to 0.75; P < .001). After a median follow-up of 31 months, median OS was 18.9 months for capecitabine and was 16.4 months for CBM; these data were not significantly different. Toxicity rates were acceptable, and all treatment regimens well tolerated. Bevacizumab toxicities were similar to those in previous studies. Measures of overall QOL were similar in all groups.
Adding bevacizumab to capecitabine, with or without mitomycin, significantly improves PFS without major additional toxicity or impairment of QOL.
Alkaptonuria (AKU) is characterised by increased circulating homogentisic acid and deposition of ochronotic pigment in collagen-rich connective tissues (ochronosis), stiffening the tissue. This ...process over many years leads to a painful and severe osteoarthropathy, particularly affecting the cartilage of the spine and large weight bearing joints. Evidence in human AKU tissue suggests that pigment binds to collagen. The exposed collagen hypothesis suggests that collagen is initially protected from ochronosis, and that ageing and mechanical loading causes loss of protective molecules, allowing pigment binding. Schmorl’s staining has previously demonstrated knee joint ochronosis in AKU mice. This study documents more comprehensively the anatomical distribution of ochronosis in two AKU mouse models (BALB/c
Hgd
−/−
,
Hgd tm1a
−/−
), using Schmorl’s staining. Progression of knee joint pigmentation with age in the two AKU mouse models was comparable. Within the knee, hip, shoulder, elbow and wrist joints, pigmentation was associated with chondrons of calcified cartilage. Pigmented chondrons were identified in calcified endplates of intervertebral discs and the calcified knee joint meniscus, suggesting that calcified tissues are more susceptible to pigmentation. There were significantly more pigmented chondrons in lumbar versus tail intervertebral disc endplates (
p
= 0.002) and clusters of pigmented chondrons were observed at the insertions of ligaments and tendons. These observations suggest that loading/strain may be associated with increased pigmentation but needs further experimental investigation. The calcified cartilage may be the first joint tissue to acquire matrix damage, most likely to collagen, through normal ageing and physiological loading, as it is the first to become susceptible to pigmentation.
Purpose
The separate impacts of dose and dose intensity of chemotherapy for metastatic breast cancer remain uncertain. The primary objective of this trial was to compare a short, high-dose, intensive ...course of epirubicin and cyclophosphamide (EC) with a longer conventional dose regimen delivering the same total dose of chemotherapy.
Methods
This open label trial randomised 235 women with metastatic breast cancer to receive either high-dose epirubicin 150 mg/m
2
and cyclophosphamide 1500 mg/m
2
with filgrastim support every 3 weeks for 3 cycles (HDEC) or standard dose epirubicin 75 mg/m
2
and cyclophosphamide 750 mg/m
2
every 3 weeks for 6 cycles (SDEC). Primary outcomes were time to progression, overall survival and quality of life.
Results
In 118 patients allocated HDEC 90% of the planned dose was delivered, compared to 96% in the 117 participants allocated SDEC. There were no significant differences in the time to disease progression (5.7 vs. 5.8 months,
P
= 0.19) or overall survival (14.5 vs. 16.5 months,
P
= 0.29) between HDEC and SDEC, respectively. Patients on HDEC reported worse quality of life during therapy, but scores improved after completion to approximate those reported by patients allocated SDEC. Objective tumour response was recorded in 33 (28%) on HDEC and 42 patients (36%) on SDEC. HDEC produced more haematologic toxicity.
Conclusion
For women with metastatic breast cancer, disease progression, survival or quality of life were no better with high-dose intensity compared to standard dose EC chemotherapy.
Australian Clinical Trials Registry registration number
ACTRN12605000478617.
Summary Background The use of prophylactic radiotherapy to prevent procedure-tract metastases (PTMs) in malignant pleural mesothelioma remains controversial, and clinical practice varies worldwide. ...We aimed to compare prophylactic radiotherapy with deferred radiotherapy (given only when a PTM developed) in a suitably powered trial. Methods We did a multicentre, open-label, phase 3, randomised controlled trial in 22 UK hospitals of patients with histocytologically proven mesothelioma who had undergone large-bore pleural interventions in the 35 days prior to recruitment. Eligible patients were randomised (1:1), using a computer-generated sequence, to receive immediate radiotherapy (21 Gy in three fractions within 42 days of the pleural intervention) or deferred radiotherapy (same dose given within 35 days of PTM diagnosis). Randomisation was minimised by histological subtype, surgical versus non-surgical procedure, and pleural procedure (indwelling pleural catheter vs other). The primary outcome was the incidence of PTM within 7 cm of the site of pleural intervention within 12 months from randomisation, assessed in the intention-to-treat population. This trial is registered with ISRCTN, number ISRCTN72767336. Findings Between Dec 23, 2011, and Aug 4, 2014, we randomised 203 patients to receive immediate radiotherapy (n=102) or deferred radiotherapy (n=101). The patients were well matched at baseline. No significant difference was seen in PTM incidence in the immediate and deferred radiotherapy groups (nine 9% vs 16 16%; odds ratio 0·51 95% CI 0·19–1·32; p=0·14). The only serious adverse event related to a PTM or radiotherapy was development of a painful PTM within the radiotherapy field that required hospital admission for symptom control in one patient who received immediate radiotherapy. Common adverse events of immediate radiotherapy were skin toxicity (grade 1 in 50 54% and grade 2 in four 4% of 92 patients vs grade 1 in three 60% and grade 2 in two 40% of five patients in the deferred radiotherapy group who received radiotherapy for a PTM) and tiredness or lethargy (36 39% in the immediate radiotherapy group vs two 40% in the deferred radiotherapy group) within 3 months of receiving radiotherapy. Interpretation Routine use of prophylactic radiotherapy in all patients with mesothelioma after large-bore thoracic interventions is not justified. Funding Research for Patient Benefit Programme from the UK National Institute for Health Research.
Alkaptonuria (AKU) is caused by homogentisate 1,2‐dioxygenase deficiency that leads to homogentisic acid (HGA) accumulation, ochronosis and severe osteoarthropathy. Recently, nitisinone treatment, ...which blocks HGA formation, has been effective in AKU patients. However, a consequence of nitisinone is elevated tyrosine that can cause keratopathy. The effect of tyrosine and phenylalanine dietary restriction was investigated in nitisinone‐treated AKU mice, and in an observational study of dietary intervention in AKU patients. Nitisinone‐treated AKU mice were fed tyrosine/phenylalanine‐free and phenylalanine‐free diets with phenylalanine supplementation in drinking water. Tyrosine metabolites were measured pre‐nitisinone, post‐nitisinone, and after dietary restriction. Subsequently an observational study was undertaken in 10 patients attending the National Alkaptonuria Centre (NAC), with tyrosine >700 μmol/L who had been advised to restrict dietary protein intake and where necessary, to use tyrosine/phenylalanine‐free amino acid supplements. Elevated tyrosine (813 μmol/L) was significantly reduced in nitisinone‐treated AKU mice fed a tyrosine/phenylalanine‐free diet in a dose responsive manner. At 3 days of restriction, tyrosine was 389.3, 274.8, and 144.3 μmol/L with decreasing phenylalanine doses. In contrast, tyrosine was not effectively reduced in mice by a phenylalanine‐free diet; at 3 days tyrosine was 757.3, 530.2, and 656.2 μmol/L, with no dose response to phenylalanine supplementation. In NAC patients, tyrosine was significantly reduced (P = .002) when restricting dietary protein alone, and when combined with tyrosine/phenylalanine‐free amino acid supplementation; 4 out of 10 patients achieved tyrosine <700 μmol/L. Tyrosine/phenylalanine dietary restriction significantly reduced nitisinone‐induced tyrosinemia in mice, with phenylalanine restriction alone proving ineffective. Similarly, protein restriction significantly reduced circulating tyrosine in AKU patients.
Time to treatment matters in traumatic haemorrhage but the optimal prehospital use of blood in major trauma remains uncertain. We investigated whether use of packed red blood cells (PRBC) and ...lyophilised plasma (LyoPlas) was superior to use of 0·9% sodium chloride for improving tissue perfusion and reducing mortality in trauma-related haemorrhagic shock.
Resuscitation with pre-hospital blood products (RePHILL) is a multicentre, allocation concealed, open-label, parallel group, randomised, controlled, phase 3 trial done in four civilian prehospital critical care services in the UK. Adults (age ≥16 years) with trauma-related haemorrhagic shock and hypotension (defined as systolic blood pressure <90 mm Hg or absence of palpable radial pulse) were assessed for eligibility by prehospital critial care teams. Eligible participants were randomly assigned to receive either up to two units each of PRBC and LyoPlas or up to 1 L of 0·9% sodium chloride administered through the intravenous or intraosseous route. Sealed treatment packs which were identical in external appearance, containing PRBC–LyoPlas or 0·9% sodium chloride were prepared by blood banks and issued to participating sites according to a randomisation schedule prepared by the co-ordinating centre (1:1 ratio, stratified by site). The primary outcome was a composite of episode mortality or impaired lactate clearance, or both, measured in the intention-to-treat population. This study is completed and registered with ISRCTN.com, ISRCTN62326938.
From Nov 29, 2016 to Jan 2, 2021, prehospital critical care teams randomly assigned 432 participants to PRBC–LyoPlas (n=209) or to 0·9% sodium chloride (n=223). Trial recruitment was stopped before it achieved the intended sample size of 490 participants due to disruption caused by the COVID-19 pandemic. The median follow-up was 9 days (IQR 1 to 34) for participants in the PRBC–LyoPlas group and 7 days (0 to 31) for people in the 0·9% sodium chloride group. Participants were mostly white (62%) and male (82%), had a median age of 38 years (IQR 26 to 58), and were mostly involved in a road traffic collision (62%) with severe injuries (median injury severity score 36, IQR 25 to 50). Before randomisation, participants had received on average 430 mL crystalloid fluids and tranexamic acid (90%). The composite primary outcome occurred in 128 (64%) of 199 participants randomly assigned to PRBC–LyoPlas and 136 (65%) of 210 randomly assigned to 0·9% sodium chloride (adjusted risk difference –0·025% 95% CI –9·0 to 9·0, p=0·996). The rates of transfusion-related complications in the first 24 h after ED arrival were similar across treatment groups (PRBC–LyoPlas 11 7% of 148 compared with 0·9% sodium chloride nine 7% of 137, adjusted relative risk 1·05 95% CI 0·46–2·42). Serious adverse events included acute respiratory distress syndrome in nine (6%) of 142 patients in the PRBC–LyoPlas group and three (2%) of 130 in 0·9% sodium chloride group, and two other unexpected serious adverse events, one in the PRBC-LyoPlas (cerebral infarct) and one in the 0·9% sodium chloride group (abnormal liver function test). There were no treatment-related deaths.
The trial did not show that prehospital PRBC–LyoPlas resuscitation was superior to 0·9% sodium chloride for adult patients with trauma related haemorrhagic shock. Further research is required to identify the characteristics of patients who might benefit from prehospital transfusion and to identify the optimal outcomes for transfusion trials in major trauma. The decision to commit to routine prehospital transfusion will require careful consideration by all stakeholders.
National Institute for Health Research Efficacy and Mechanism Evaluation.
Copy number variants (CNVs) account for a major proportion of human genetic polymorphism and have been predicted to have an important role in genetic susceptibility to common disease. To address this ...we undertook a large, direct genome-wide study of association between CNVs and eight common human diseases. Using a purpose-designed array we typed approximately 19,000 individuals into distinct copy-number classes at 3,432 polymorphic CNVs, including an estimated approximately 50% of all common CNVs larger than 500 base pairs. We identified several biological artefacts that lead to false-positive associations, including systematic CNV differences between DNAs derived from blood and cell lines. Association testing and follow-up replication analyses confirmed three loci where CNVs were associated with disease-IRGM for Crohn's disease, HLA for Crohn's disease, rheumatoid arthritis and type 1 diabetes, and TSPAN8 for type 2 diabetes-although in each case the locus had previously been identified in single nucleotide polymorphism (SNP)-based studies, reflecting our observation that most common CNVs that are well-typed on our array are well tagged by SNPs and so have been indirectly explored through SNP studies. We conclude that common CNVs that can be typed on existing platforms are unlikely to contribute greatly to the genetic basis of common human diseases.
Metabolomic analyses in alkaptonuria (AKU) have recently revealed alternative pathways in phenylalanine-tyrosine (phe-tyr) metabolism from biotransformation of homogentisic acid (HGA), the active ...molecule in this disease. The aim of this research was to study the phe-tyr metabolic pathway and whether the metabolites upstream of HGA, increased in nitisinone-treated patients, also undergo phase 1 and 2 biotransformation reactions. Metabolomic analyses were performed on serum and urine from patients partaking in the SONIA 2 phase 3 international randomised-controlled trial of nitisinone in AKU (EudraCT no. 2013-001633-41). Serum and urine samples were taken from the same patients at baseline (pre-nitisinone) then at 24 and 48 months on nitisinone treatment (patients N = 47 serum; 53 urine) or no treatment (patients N = 45 serum; 50 urine). Targeted feature extraction was performed to specifically mine data for the entire complement of theoretically predicted phase 1 and 2 biotransformation products derived from phenylalanine, tyrosine, 4-hydroxyphenylpyruvic acid and 4-hydroxyphenyllactic acid, in addition to phenylalanine-derived metabolites with known increases in phenylketonuria. In total, we observed 13 phase 1 and 2 biotransformation products from phenylalanine through to HGA. Each of these products were observed in urine and two were detected in serum. The derivatives of the metabolites upstream of HGA were markedly increased in urine of nitisinone-treated patients (fold change 1.2–16.2) and increases in 12 of these compounds were directly proportional to the degree of nitisinone-induced hypertyrosinaemia (correlation coefficient with serum tyrosine = 0.2–0.7). Increases in the urinary phenylalanine metabolites were also observed across consecutive visits in the treated group. Nitisinone treatment results in marked increases in a wider network of phe-tyr metabolites than shown before. This network comprises alternative biotransformation products from the major metabolites of this pathway, produced by reactions including hydration (phase 1) and bioconjugation (phase 2) of acetyl, methyl, acetylcysteine, glucuronide, glycine and sulfate groups. We propose that these alternative routes of phe-tyr metabolism, predominantly in urine, minimise tyrosinaemia as well as phenylalanaemia.
Background: We aimed to study fecal calprotectin in Scottish children with inflammatory bowel disease (IBD) and compare its diagnostic accuracy with blood parameters.
Methods: Stool samples from 48 ...Scottish children (29 males, 19 females) had calprotectin measured at IBD diagnosis. The median age at diagnosis was 11.2 years (interquartile range IQR 8.7–13.0 years). There were 33 patients with Crohn's disease, 5 with ulcerative colitis, and 10 with IBD type unspecified. IBD was diagnosed by standard criteria. Calprotectin was measured using a commercially available kit (PhiCal Test) and 47/48 patients had comparative blood results available at diagnosis.
Results: The fecal calprotectin concentrations were raised in 96% (46/48) of patients studied. The median calprotectin value was 750 μg/g (IQR 235.8–1251 μg/g). In comparison with standard blood tests, 32/45 (71.1%) had abnormal erythrocyte sedimentation rate, 19/38 (50.0%) had abnormal C‐reactive protein, 29/46 (63.0%) had raised platelets, 12/45 (26.7%) had hypoalbuminemia, and 38/46 (82.6%) had abnormal hemoglobin. We identified 7/47 (14.9%) patients with raised calprotectin at diagnosis who did not have any abnormalities detected in the blood tests performed. All 48 patients (100%) had at least 1 abnormal blood test and/or raised calprotectin at diagnosis.
Conclusions: Calprotectin is significantly more likely to be raised than any commonly employed blood tests at IBD diagnosis. When used in combination with these bloods tests an abnormality was demonstrated in 1 or both tests in all patients at diagnosis in this study. Fecal calprotectin measurement is a significant advance when used contemporaneously and in addition to a routine panel of blood tests in the diagnosis of pediatric IBD.
(Inflamm Bowel Dis 2008)
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