A partial human LCK defect causes a T cell immunodeficiency with intestinal inflammation.

Lymphocyte-specific protein tyrosine kinase (LCK) is essential for T cell antigen receptor (TCR)-mediated signal transduction. Here, we report two siblings homozygous for a novel LCK variant (c.1318C>T; P440S) characterized by T cell lymphopenia with skewed memory phenotype, infant-onset recurrent infections, failure to thrive, and protracted diarrhea. The patients' T cells show residual TCR signal transduction and proliferation following anti-CD3/CD28 and phytohemagglutinin (PHA) stimulation. We demonstrate in mouse models that complete (Lck-/-) versus partial (LckP440S/P440S) loss-of-function LCK causes disease with differing phenotypes. While both Lck-/- and LckP440S/P440S mice exhibit arrested thymic T cell development and profound T cell lymphopenia, only LckP440S/P440S mice show residual T cell proliferation, cytokine production, and intestinal inflammation. Furthermore, the intestinal disease in the LckP440S/P440S mice is prevented by CD4+ T cell depletion or regulatory T cell transfer. These findings demonstrate that P440S LCK spares sufficient T cell function to allow the maturation of some conventional T cells but not regulatory T cells-leading to intestinal inflammation.

Dysregulated Lymphocyte Antigen Receptor Signaling in Common Variable Immunodeficiency with Granulomatous Lymphocytic Interstitial Lung Disease.

PURPOSE: A subset of common variable immunodeficiency (CVID) patients either presents with or develops autoimmune and lymphoproliferative complications, such as granulomatous lymphocytic interstitial lung disease (GLILD), a major cause of morbidity and mortality in CVID. While a myriad of phenotypic lymphocyte derangements has been associated with and described in GLILD, defects in T and B cell antigen receptor (TCR/BCR) signaling in CVID and CVID with GLILD (CVID/GLILD) remain undefined, hindering discovery of biomarkers for disease monitoring, prognostic prediction, and personalized medicine approaches. METHODS: To identify perturbations of immune cell subsets and TCR/BCR signal transduction, we applied mass cytometry analysis to peripheral blood mononuclear cells (PBMCs) from healthy control participants (HC), CVID, and CVID/GLILD patients. RESULTS: Patients with CVID, regardless of GLILD status, had increased frequency of HLADR+CD4+ T cells, CD57+CD8+ T cells, and CD21lo B cells when compared to healthy controls. Within these cellular populations in CVID/GLILD patients only, engagement of T or B cell antigen receptors resulted in discordant downstream signaling responses compared to CVID. In CVID/GLILD patients, CD21lo B cells showed perturbed BCR-mediated phospholipase C gamma and extracellular signal-regulated kinase activation, while HLADR+CD4+ T cells and CD57+CD8+ T cells displayed disrupted TCR-mediated activation of kinases most proximal to the receptor. CONCLUSION: Both CVID and CVID/GLILD patients demonstrate an activated T and B cell phenotype compared to HC. However, only CVID/GLILD patients exhibit altered TCR/BCR signaling in the activated lymphocyte subsets. These findings contribute to our understanding of the mechanisms of immune dysregulation in CVID with GLILD.

Formin-like 1 mediates effector T cell trafficking to inflammatory sites to enable T cell-mediated autoimmunity.

Lymphocyte migration is essential for the function of the adaptive immune system, and regulation of T cell entry into tissues is an effective therapy in autoimmune diseases. Little is known about the specific role of cytoskeletal effectors that mediate mechanical forces and morphological changes essential for migration in complex environments. We developed a new Formin-like-1 (FMNL1) knock-out mouse model and determined that the cytoskeletal effector FMNL1 is selectively required for effector T cell trafficking to inflamed tissues, without affecting naïve T cell entry into secondary lymphoid organs. Here, we identify a FMNL1-dependent mechanism of actin polymerization at the back of the cell that enables migration of the rigid lymphocyte nucleus through restrictive barriers. Furthermore, FMNL1-deficiency impairs the ability of self-reactive effector T cells to induce autoimmune disease. Overall, our data suggest that FMNL1 may be a potential therapeutic target to specifically modulate T cell trafficking to inflammatory sites.

Molecular Basis of Valine-Citrulline-PABC Linker Instability in Site-Specific ADCs and Its Mitigation by Linker Design.

The degree of stability of antibody-drug linkers in systemic circulation, and the rate of their intracellular processing within target cancer cells are among the key factors determining the efficacy of antibody-drug conjugates (ADC) in vivo Previous studies demonstrated the susceptibility of cleavable linkers, as well as auristatin-based payloads, to enzymatic cleavage in rodent plasma. Here, we identify Carboxylesterase 1C as the enzyme responsible for the extracellular hydrolysis of valine-citrulline-p-aminocarbamate (VC-PABC)-based linkers in mouse plasma. We further show that the activity of Carboxylesterase 1C towards VC-PABC-based linkers, and consequently the stability of ADCs in mouse plasma, can be effectively modulated by small chemical modifications to the linker. While the introduced modifications can protect the VC-PABC-based linkers from extracellular cleavage, they do not significantly alter the intracellular linker processing by the lysosomal protease Cathepsin B. The distinct substrate preference of the serum Carboxylesterase 1C offers the opportunity to modulate the extracellular stability of cleavable ADCs without diminishing the intracellular payload release required for ADC efficacy. Mol Cancer Ther; 15(5); 958-70. ©2016 AACR.

Production of soluble and active microbial transglutaminase in Escherichia coli for site-specific antibody drug conjugation.

Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro-domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine-scan of the mTGase pro-domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro-domain/mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 20°C, but results in full activity when shifted to 37°C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro-domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30-75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site-specific antibody drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.

Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy.

The efficacy of an antibody-drug conjugate (ADC) is dependent on the properties of its linker-payload which must remain stable while in systemic circulation but undergo efficient processing upon internalization into target cells. Here, we examine the stability of a non-cleavable Amino-PEG6-based linker bearing the monomethyl auristatin D (MMAD) payload site-specifically conjugated at multiple positions on an antibody. Enzymatic conjugation with transglutaminase allows us to create a stable amide linkage that remains intact across all tested conjugation sites on the antibody, and provides us with an opportunity to examine the stability of the auristatin payload itself. We report a position-dependent degradation of the C terminus of MMAD in rodent plasma that has a detrimental effect on its potency. The MMAD cleavage can be eliminated by either modifying the C terminus of the toxin, or by selection of conjugation site. Both approaches result in improved stability and potency in vitro and in vivo. Furthermore, we show that the MMAD metabolism in mouse plasma is likely mediated by a serine-based hydrolase, appears much less pronounced in rat, and was not detected in cynomolgus monkey or human plasma. Clarifying these species differences and controlling toxin degradation to optimize ADC stability in rodents is essential to make the best ADC selection from preclinical models. The data presented here demonstrate that site selection and toxin susceptibility to mouse plasma degradation are important considerations in the design of non-cleavable ADCs, and further highlight the benefits of site-specific conjugation methods.

Effect of attachment site on stability of cleavable antibody drug conjugates.

The systemic stability of the antibody-drug linker is crucial for delivery of an intact antibody-drug conjugate (ADC) to target-expressing tumors. Linkers stable in circulation but readily processed in the target cell are necessary for both safety and potency of the delivered conjugate. Here, we report a range of stabilities for an auristatin-based payload site-specifically attached through a cleavable valine-citrulline-p-aminobenzylcarbamate (VC-PABC) linker across various sites on an antibody. We demonstrate that the conjugation site plays an important role in determining VC-PABC linker stability in mouse plasma, and that the stability of the linker positively correlates with ADC cytotoxic potency both in vitro and in vivo. Furthermore, we show that the VC-PABC cleavage in mouse plasma is not mediated by Cathepsin B, the protease thought to be primarily responsible for linker processing in the lysosomal degradation pathway. Although the VC-PABC cleavage is not detected in primate plasma in vitro, linker stabilization in the mouse is an essential prerequisite for designing successful efficacy and safety studies in rodents during preclinical stages of ADC programs. The divergence of linker metabolism in mouse plasma and its intracellular cleavage offers an opportunity for linker optimization in the circulation without compromising its efficient payload release in the target cell.

The principle of assessing mental capacity for enduring power of attorney.

With Hong Kong's rapidly ageing population, increasing numbers of people now have some form of cognitive impairment. Enduring power of attorney is a legal instrument that can allow individuals to manage their financial matters if they subsequently become mentally incapacitated. The law requires that the mental capacity of the individual making an enduring power of attorney should be certified by a registered medical practitioner and a solicitor. This paper discusses the principles involved in the assessment of mental capacity for making an enduring power of attorney and uses this example to illustrate various important considerations in the formal assessment of mental capacity.

Structured assessment of mental capacity to make financial decisions in Chinese older persons with mild cognitive impairment and mild Alzheimer disease.

Previous studies suggested that patients with mild cognitive impairment (MCI) or dementia can have impaired and declining financial skills and abilities. The purpose of this study is to test a clinically applicable method, based on the contemporary legal standard, to examine directly the mental capacity to make financial decisions and its component decision-making abilities among patients with MCI and early dementia. A total of 90 patients with mild Alzheimer disease (AD), 92 participants with MCI, and 93 cognitively normal control participants were recruited for this study. Their mental capacity to make everyday financial decisions was assessed by clinician ratings and the Chinese version of the Assessment of Capacity for Everyday Decision-Making (ACED). Based on the clinician ratings, only 53.5% were found to be mentally competent in the AD group, compared with 94.6% in the MCI group. However, participants with MCI had mild but significant impairment in understanding, appreciating, and reasoning abilities as measured by the ACED. The ACED provided a reliable and clinically applicable structured framework for assessment of mental capacity to make financial decisions.

Capacity to make decisions on medication management in Chinese older persons with mild cognitive impairment and mild Alzheimer's disease.

BACKGROUND: This study aimed to assess if decisional capacity and the four decision-making abilities related to decisions concerning medication management were impaired among community-dwelling Chinese older persons in Hong Kong with amnestic mild cognitive impairment (MCI) and mild Alzheimer's disease (AD), as compared with cognitively normal older adults. METHODS: Two hundred and ninety-one Chinese community-dwelling older adults were recruited. The four decision-making abilities and decisional capacity were assessed by using the Chinese version of the Assessment of Capacity for Everyday Decision-Making (ACED) and independent clinician ratings based on the definition in the UK Mental Capacity Act 2005, respectively. RESULTS: Ninety-nine participants (34%) were diagnosed with MCI and ninety-five (33%) with mild AD. Although almost all (96%) of the participants in the MCI group were found to be mentally competent to make decisions on medication management in clinician ratings, their decision-making abilities as measured by the ACED were significantly lower than those of the cognitively normal controls. CONCLUSIONS: Results from this study suggest that abilities related to decisions on medication management are impaired before the clinical diagnosis of dementia is made. Use of specific and structured assessment of the relevant decisional abilities may enhance clinical judgment.

A PIN1 polymorphism that prevents its suppression by AP4 associates with delayed onset of Alzheimer's disease.

Alzheimer's disease (AD), the most common form of dementia, is characterized by the presence of neurofibrillary tangles composed of tau and senile plaques of amyloid-beta peptides (Aß) derived from amyloid precursor protein (APP). Pin1 is a unique prolyl isomerase that has been shown to protect against age-dependent neurodegeneration by acting on phosphorylated tau and APP to suppress tangle formation and amyloidogenic APP processing. Here we report a functional polymorphism, rs2287839, in the Pin1 promoter that is significantly associated with a 3-year delay in the average age at onset (AAO) of late-onset AD in a Chinese population. More significantly, the Pin1 polymorphism rs2287839 is located within the consensus binding motif for the brain-selective transcription factor, AP4 (CAGCTG) and almost completely abolishes the ability of AP4 to bind and suppress the Pin1 promoter, as shown by chromatin immunoprecipitation, electrophoretic mobility shift assay, and promoter luciferase assay. Moreover, overexpression or knockdown of AP4 resulted in an 80% reduction or 2-fold increase in endogenous Pin1 levels, respectively. Thus, AP4 is a novel transcriptional repressor of Pin1 expression and the Pin1 promoter single nucleotide polymorphism (SNP) identified in this study that prevents such suppression is associated with delayed onset of AD. These results indicate that regulation of Pin1 by AP4 plays a critical role in determining age at onset of AD and might be a novel therapeutic target to delay the onset of AD.

NEDD9 gene polymorphism influences the risk of Alzheimer disease and cognitive function in Chinese older persons.

Neural precursor cell expressed, developmentally down-regulated (NEDD9) gene was a new candidate risk gene for Alzheimer disease (AD). The CC genotype of a single nucleotide polymorphism rs760678 within this gene was associated with increasing risk of AD in a large study with white population. Our study aimed to replicate the initial report in Chinese population and explore its effect on cognitive performance. A total of 262 patients with AD, 293 patients with mild cognitive impairment, and 434 cognitive intact controls were recruited in the study. The result showed that G allele had a greater risk of AD (χ for trend test=5.61, df 1, P=0.018). The effects were mainly observed among Apolipoprotein E (APOE) ε4 noncarriers (χ for trend test=4.30, df 1, P=0.038). After adjustment of sex, age, education year, and APOE ε4 status by logistic regression, significant association between NEDD9 GG genotype and AD remained [OR=2.04, 95% confidence interval (CI)=1.02-4.08, P=0.044]. The scores of Cantonese version of the Mini-mental State Examination and Alzheimer's Disease Assessment Subscale-Cognitive subscale were associated with N polymorphism after adjusting for sex, age, education year, and ApoE ε4 status (Linear regression model, P<0.05). Our study identified rs760678 within NEDD9 gene in association with the risk of AD and cognitive performance in Chinese older persons. The fact that different alleles accounted for the risk in different population might suggest that there were ethnic group specific haplotypes that were primarily responsible for the predisposition.

Interim follow-up of a randomized controlled trial comparing Chinese style mind body (Tai Chi) and stretching exercises on cognitive function in subjects at risk of progressive cognitive decline.

OBJECTIVES: We reported the interim findings of a randomized controlled trial (RCT) to examine the effects of a mind body physical exercise (Tai Chi) on cognitive function in Chinese subjects at risk of cognitive decline. SUBJECTS: 389 Chinese older persons with either a Clinical Dementia Rating (CDR 0.5) or amnestic-MCI participated in an exercise program. The exercise intervention lasted for 1 year; 171 subjects were trained with 24 forms simplified Tai Chi (Intervention, I) and 218 were trained with stretching and toning exercise (Control, C). The exercise comprised of advised exercise sessions of at least three times per week. RESULTS: At 5th months (2 months after completion of training), both I and C subjects showed an improvement in global cognitive function, delayed recall and subjective cognitive complaints (paired t-tests, p < 0.05). Improvements in visual spans and CDR sum of boxes scores were observed in I group (paired t-tests, p < 0.001). Three (2.2%) and 21(10.8%) subjects from the I and C groups progressed to dementia (Pearson chi square = 8.71, OR = 5.34, 95% CI 1.56-18.29). Logistic regression analysis controlled for baseline group differences in education and cognitive function suggested I group was associated with stable CDR (OR = 0.14, 95%CI = 0.03-0.71, p = 0.02). CONCLUSIONS: Our interim findings showed that Chinese style mind body (Tai Chi) exercise may offer specific benefits to cognition, potential clinical interests should be further explored with longer observation period.

Neuropsychiatric symptoms are associated with increased risks of progression to dementia: a 2-year prospective study of 321 Chinese older persons with mild cognitive impairment.

BACKGROUND: studies have suggested that neuropsychiatric (NP) symptoms influence the development of dementia among older adults. But, the results are inconsistent and there is limited information about NP symptoms in population-based samples. OBJECTIVE: to explore the association between NP symptoms and risk of cognitive decline in Chinese older persons residing in the community. DESIGN: prospective study. SETTING: community sample. SUBJECTS: a total of 321 community-dwelling Chinese older persons aged 60 or over with mild cognitive impairment participated in the study. METHODS: at baseline, each subject was assessed with Clinical Dementia Rating (CDR), Mini-Mental State Examination, list learning and delayed recall, and Category Verbal Fluency Test. Severity of NP symptoms was evaluated with Neuropsychiatric Inventory (NPI). Global cognitive status at the end of 2-year study period was determined by CDR. RESULTS: at baseline, 40.5% of participants exhibited one or more NP symptoms (NPI total score ≥ 1). Night-time behaviours (22.1%), depression (16.8%), apathy (14.0%) and anxiety (12.8%) were the most common NP symptoms. At the end of 2-year follow-up, 27.5% of participants with depression at baseline developed dementia, compared with 14.8% of those without depression (χ² = 4.90, P= 0.03). Aberrant motor behaviour was also significantly associated with deterioration in cognition (χ² = 5.84, P= 0.02), although it was an infrequent occurrence. On logistic regression analysis, only depression at baseline was shown to be a risk factor for progression to dementia (OR= 2.40, 95% CI 1.05-5.46, P= 0.04). CONCLUSION: depression in non-demented older persons may represent an independent dimension reflecting early neuronal degeneration. Further studies should be conducted to assess whether effective management of NP symptoms exerts beneficial effects on cognitive function.

Examining the association between late-life leisure activity participation and global cognitive decline in community-dwelling elderly Chinese in Hong Kong.

OBJECTIVE: This study examines the association between late-life leisure activity participation and global cognitive decline in community-dwelling elderly Chinese in Hong Kong. METHODS: Five hundred and five participants, not clinically demented at the baseline, were analysed in the follow-up study of a population-based community survey among Hong Kong Chinese aged 60 and over. Information regarding leisure activity participation, global cognitive function and important sociodemographic variables was collected. Late life leisure activity profiles were classified into intellectual, social, physical and recreational categories, and were measured by total hours per week, total frequency and total number of subtypes. Multivariate logistic regression analyses were used to evaluate the association between leisure activity participation at the baseline and the incidence of global cognitive decline at the 22-month follow-up. The incidence of global cognitive decline was defined as a one-point drop in z-score of the Cantonese version of the mini-mental state examination (CMMSE). RESULTS: At the follow-up, a higher level of participation in intellectual activities was significantly associated with a lower incidence of global cognitive decline as measured by both the total hours per week (multivariate-adjusted OR 0.97 (95% CI 0.94-0.99, p=0.003)), and the total number of subtypes (multivariate-adjusted OR 0.74 (95% CI 0.58-0.95, p=0.018)). CONCLUSIONS: A higher level of late-life intellectual activity participation was associated with less global cognitive decline among community-dwelling elderly Chinese in Hong Kong.

Could hypothetical scenarios enhance understanding on decision for life-sustaining treatment in non-demented Chinese older persons?

BACKGROUND: With increasing longevity, there is an increasing need for medical professionals to face situations in which explanation for decision on life-sustaining treatment (LST) would be required. OBJECTIVES: As advance decision making for LST in case of severe medical illness may be unfamiliar for most of the Chinese elders, we aim to explore if procedures adopted to enhance the exposure to the issue concerned would bring about improvement in knowledge toward decision for LST. METHOD: This was a cross-sectional study. The design was divided into three sections: (i) a pre-scenario knowledge assessment, (ii) scenario exposure (relating issues of LST using case vignettes), and (iii) a post-scenario assessment. The pre- and post-scenario assessment comprises 10 questions, exploring the understanding toward basic issues related to LST. The scenario exposure comprises two hypothetical case vignettes describing situations demanding decisions for LST. The knowledge level toward LST was assessed and compared before and after the presentation of the two vignettes. RESULTS: One-hundred community dwelling older persons (aged over 60 years) were recruited. The scenario exposure improved the knowledge level of participants (paired samples t-test, p < 0.05). Participants who were younger and better educated were more likely to perform better in the knowledge test (bivariate correlation and logistic regression, p < 0.05). CONCLUSIONS: The results demonstrated that hypothetical scenarios may help to enhance and facilitate the understanding of LST. The study should be carried forward to explore the applicability of enhancement procedure to facilitate the decision making for advance directives and LST in the older community.

Prevalence of neuropsychiatric symptoms in chinese older persons with mild cognitive impairment-a population-based study.

OBJECTIVES: To estimate the point prevalence and correlates of neuropsychiatric (NP) symptoms among older adults with mild cognitive impairment (MCI) and normal cognition (NC) in a Chinese community. DESIGN: Cross-sectional study derived from a population-based prevalence study of MCI and dementia. SETTING AND PARTICIPANTS: This survey was conducted in Hong Kong from 2005 to 2006. Seven hundred eighty-eight community-dwelling older adults (450 NC and 338 MCI) were recruited. Cognitive and NP data were obtained. RESULTS: The point prevalence of at least one NP symptom in NC and MCI were 29% and 36.7%, respectively (logistic regression controlled for age and education, odds ratio = 1.38, 95% confidence interval [CI]: 1.01-1.89, Wald χ = 4.10, df = 1, p = 0.04). Agitation (1.8% versus 5.1%), apathy (7.6% versus 15.2%), and irritability (4.2% versus 8%) were more prevalent in subjects with MCI (p <0.05). Logistic regression analyses showed that apathy score was a significant factor associated with the status of NC or MCI (logistic regression, apathy, p = 0.031, Exp(B) = 1.23, 95% CI: 1.02-1.47; Hosmer and Lemeshow test, χ = 8.6, df = 8, p = 0.38, R = 0.23). CONCLUSIONS: The authors reported the findings of one of the first population-based studies estimating the point prevalence of NP symptoms in Asian older adults with MCI. Taking into account of its prevalence and magnitude of effects, apathy is a clinically significant symptom in MCI. Its predictive value for conversion to dementia warrants further evaluation.

Combined clinical and cognitive criteria to identify mild cognitive impairment in a southern Chinese community.

Mild Cognitive Impairment (MCI) is a recognized risk condition for clinical dementia. This paper attempted to explore the applicability of a combined cognitive and clinical approach to identify older Chinese adults at-risk of cognitive decline. Seven hundred forty randomly recruited community dwelling participants (aged 60 or over) were assessed at baseline and 2 years with Clinical Dementia Rating (CDR) and a cognitive battery. Baseline MCI groups were categorized by CDR-MCI, cognitive function (Cog-MCI), and a combined CDR-Cog approach. The cognitive approach adopted the Mayo clinic criteria. For the combined approach, nonamnestic MCI combined CDR 0.5 plus nonmemory cognitive deficits. The overall concordance between CDR and Cognitive test ratings were 65.3% (χ2 = 256.4, P<0.001, κ=0.44). With a combined approach, 424 (57%) participants were classified as normal. CDR-MCI group had higher cognitive scores compared with MCI groups by other criteria (1 way analysis of variance or ANOVA). At 2 years, the combined CDR-Cog MCI group identified all dementia (N=24) converters although group differences were not significant. Cognitive function and CDR identified participants potentially at-risk for furthermore decline, but exhibited some differences in detection profiles. A combined approach may be more practical in screening for MCI participants with diverse educational and cultural background.

Serum zinc is decreased in Alzheimer's disease and serum arsenic correlates positively with cognitive ability.

Zinc, copper, and iron aggregate Abeta and accumulate in Alzheimer's disease (AD) plaques. Some metals are increased in AD vs. control serum. The authors examined levels of 12 metals in serum of 44 AD and 41 control subjects. Zinc decreased from 12.3 to 10.9 micromol/L (means, p = 0.0007). Arsenic positively correlated with Mini-Mental State Examination score (p < 0.0001). Zinc deposition in brain amyloid might deplete zinc from other body compartments, such as serum. The arsenic correlation might be caused by the major contribution of seafood consumption to intake of both arsenic and docosahexaenoic acid, of which the latter may delay AD.