Despite advances in supportive care, moderate-severe acute respiratory distress syndrome (ARDS) is associated with high mortality rates, and novel therapies to treat this condition are needed. Compelling pre-clinical data from mouse, rat, sheep and ex vivo perfused human lung models support the use of human mesenchymal stem  cells (MSCs) as a novel intravenous therapy for the early treatment of ARDS.This article describes the study design and challenges encountered during the implementation and phase 1 component of the START (STem cells for ARDS Treatment) trial, a phase 1/2 trial of bone marrow-derived human MSCs for moderate-severe ARDS. A trial enrolling 69 subjects is planned .This report describes study design features that are unique to a phase 1 trial in critically ill subjects and the specific challenges of implementation of a cell-based therapy trial in the ICU.Experience gained during the design and implementation of the START study will be useful to investigators planning future phase 1 clinical trials based in the ICU, as well as trials of cell-based therapy for other acute illnesses.NCT01775774 and NCT02097641.Clinical Trials Registration:  At present, the mortality rate of severe ARDS remains unacceptably high, in the range of 30 to 40% [Morbidity and mortality have declined only modestly in patients with the acute respiratory distress syndrome (ARDS) in the last decade despite extensive research into its pathophysiology -3]. Cur. Cur3]. 0 to 40% ,7]. The. Thein vCell-based therapy with human mesenchymal stem/stromal cells (MSCs) is attractive as a potential new treatment for ARDS for multiple reasons. MSCs have the ability to secrete multiple paracrine factors, such as growth factors that can enhance tissue repair, anti-inflammatory cytokines that can reduce inflammation, and antimicrobial peptides that can reduce the severity of bacterial infection -17]. MS. MS17]. ex vivo perfused human lung model, have demonstrated the potential efficacy and safety of administering human MSCs for the treatment of ARDS ,11],[11]ex v,[11]ex v of ARDS ,14]. Ba. Baex vi2/FiO2 ratio of less than 200 mmHg while on positive pressure ventilation with at least 8 cmH2O positive end-expiratory pressure (PEEP) ,,..2/FiO2, Systemic efficacy endpoints will include the mean Sequential Organ Failure Assessment (SOFA) score  at day 3Biological endpoints will focus on the proposed mechanisms of action of the human MSCs in ARDS based on preclinical studies. Specifically, we will measure plasma markers of lung epithelial injury (Receptor for Advanced Glycation Endproducts (RAGE)), pro- and anti-inflammatory markers , markers of endothelial injury , markers of other organ injury (creatinine) and biomarkers that may reflect the paracrine activity of the administered human MSCs (angiopoeitin-1 and keratinocyte growth factor). All of these biomarkers will be measured at baseline and day 3 using enzyme-linked immunoassays (ELISAs). In the phase 2 study, we plan to measure a marker of lung epithelial permeability   as well 2/FiO2 ratio < 200 with at least 8 cmH2O positive end-expiratory airway pressure (PEEP), bilateral infiltrates consistent with pulmonary edema on frontal chest radiograph and no clinical evidence of left atrial hypertension for the bilateral pulmonary infiltrates. To avoid enrolling patients with late ARDS, patients must be enrolled and randomized within 96 hours of meeting the Berlin definition for ARDS  infusionIn the supine position, patients must sustain:1. Transcutaneous oxygen saturation in the target range of 88 to 95% without any increase in ventilator settings2. Stable vasopressor use if the patient requires vasopressors for blood pressure support. The dose of vasopressor may be able to be increased no more than: 5 mcg/minute for norepinephrine 50 mcg/minute increase for phenylephrine dose 5 mcg/kg/minute increase for dopamine dose 0.5 mcg/kg/minute increase for epinephrine.These criteria were designed to reduce noise such that a harmful effect of MSCs could be more clearly identified. Cell-based therapy requires coordination with the bone marrow transplantation facility for investigational product preparation. Patients in phase 2 will be randomized after confirmation that the cells can be prepared and delivered to the ICU within four hours and when patients are likely to achieve a two-hour stable baseline period.6 cells/kg, 5 × 106 cells/kg, and 10 × 106 cells/kg predicted body weight in an escalating fashion. In phase 2, subjects will be randomized in a 2:1 randomization scheme to receive the maximum tolerable dose of MSCs determined in phase 1 or Plasma-lyte A placebo. Sixty patients will be randomized by bone marrow transplant laboratory personnel using a centralized data management system. Blinding of the investigational product will be preserved throughout the study. No treatment group information is provided to the investigators or clinicians caring for the patient except in case of an emergency, and a log of unblinding events will be maintained. An unaffiliated external medical monitor will work with investigators to determine when and if unblinding should occur.In phase 1, there are three planned cohorts with 3 subjects in each cohort who will receive doses of 1 × 10All study procedures except for pre-screening occur after informed consent is obtained from a subject or his/her surrogate. The study flow is summarized in Figure 2 ≥ 0.9, PEEP ≥15 cm H2O, 3 vasopressors or the use of > 0.1 mcg/kg/minute epinephrine for blood pressure support. Finally, given the recent interest in prone positioning, we amended the protocol to state explicitly that patients must be in the supine position for the baseline period, cell infusion and post-infusion monitoring period to reduce the likelihood of respiratory or hemodynamic instability related to changes in position.Because critically ill patients often experience minute-to-minute changes in vital signs, we have mandated that the MSCs infusion can only begin after a two-hour period of stable baseline has been documented. Stable baseline is defined as: transcutaneous oxygen saturation in the target range of 88 to 95% without any increase in ventilator settings and stable vasopressor use if the patient requires vasopressors for blood pressure support. The dose of vasopressor may be able to be increased a small amount during this two-hour period, predefined as in Table The investigational product can be infused via peripheral or central venous access, though if administered through a peripheral intravenous access, it should be at least 20-gauge, and ideally 18-gauge. The protocol recommends against co-administration with a medication or dextrose-containing solution. The cells are administered through a standard blood filter tubing set with a 170 to 260 micron filter via gravity; droplet count is used to control the infusion rate. The rationale for this approach is that several of the laboratories in our group do not routinely infuse cell-based therapies via an infusion pump, which has the potential to cause mechanical disruption of the cells. The investigational product is infused over approximately 60 minutes. Ventilator management, including weaning, follows the modified ARDS Network lower tidal volume (6 ml/kg predicted body weight) protocol because using this ventilator management protocol will standardize the application of PEEP, which is a component of one of the respiratory efficacy endpoints, the LIS, thus reducing the potential for bias. Patients are managed with a conservative fluid management protocol. The fluid management strategy is held for four hours prior to and six hours after the MSC infusion to reduce the likelihood that the fluid management strategy impacts hemodynamic stability around the time of the study intervention.Blood and urine samples are obtained at multiple time points before and after the infusion of investigational product for biomarker measurements, which include measurements of epithelial injury, inflammation and of MSC administration. In the phase 2 component, a mini-bronchoalveolar lavage is planned.For clinical endpoint and safety measurements, if not obtained as part of clinical care, patients will undergo an arterial blood gas measurement and chest radiograph on day 3  since these are secondary endpoints of the phase 2 trial. Serum creatinine, total bilirubin and alanine aminotransferase (ALT) are measured on days 3, 7 and 14  for safety monitoring if subjects are still hospitalized. Vital status as well as the need for dialysis will be assessed at 6 and 12 months after study enrollment via a telephone visit and in-person visit, respectively. Data on subsequent hospitalizations will also be collected. At the in-person visit, a limited physical exam will be conducted.At the time of cell thaw at each clinical site, samples of the MSCs are taken by the bone marrow transplantation laboratory for standardized quantitation of cell viability and for cell sterility assays. Protocols for cell thawing and washing as well as infusion were developed at the coordinating center at the University of California, San Francisco. These include the use of standard calculations to determine the appropriate dose of MSCs, standard protocols for cell thaw, transfer and washing, as well as protocols to ensure that the infusion is delivered over the protocol-specified timeframe. These standard operating procedures were adapted at each clinical site and reviewed and approved by the Coordinating Center prior to initiation of study enrollment at each site. Given the cell-based nature of this therapy and concerns about cell viability with variability in cell handling, this is a critical step for the implementation of a cell-based therapy study.Detailed demographic data and medical history, including smoking and alcohol history and baseline renal function, as well as data for baseline severity of illness, including the Acute Physiology, Age, Chronic Health Evaluation (APACHE) III , SOFA 2, and BruP = 0.004) and smaller than that reported for higher dose methylprednisolone for late ARDS  . Ho. HoP = 0For the phase 1 study, the analysis will be descriptive. Specifically, we will describe the incidence of serious adverse events, including death, as well as the incidence of pre-specified infusion associated events and non-serious adverse events felt to be related to the study infusion.t-test. The per-treatment arm sample sizes were generated based on an assumption of a 28-day adverse event proportion of 30%. In this setting, the 95% CI length for a binomial proportion is 24%, ranging from 19% to 43%.For the phase 2 study, we will describe the incidence of serious adverse events, including death, as well as the incidence of pre-specified infusion associated events and non-serious adverse events felt to be related to the study infusion in the MSC-treated versus placebo arms. Analysis of the primary safety endpoint will be focused on characterizing the adverse event proportion in each treatment arm, whereas the secondary efficacy endpoints will be used for the design of larger efficacy studies. We will compare the incidence of adverse events by treatment cohort using the Pearson’s chi-square test or the Student’s Because the phase 2 trial is focused on safety and has limited power for physiological endpoints, the analysis will be based on an approach using standardized effect sizes . This approach allows us to evaluate the efficacy of MSCs in a small sample size by comparing the effect size observed in our phase 2 trial to effects observed in larger trials of therapies known to be efficacious. In the case of ARDS, the comparison trials would be those of lower tidal volume ventilation  and fluiAs described in the prior sections, unique features of this clinical trial include the requirement for a two-hour baseline stability period prior to investigational product infusion and the definition of pre-specified infusion associated events. These are intended to ensure subject safety despite underlying critical illness and to minimize the potential for instability due to the severity of underlying illness rather than the investigational product.The data safety and monitoring plan for this phase 1/2 trial includes a formal Data Safety Monitoring Board (DSMB) that has been reviewed and approved by the IRB at the University of California, San Francisco and the National Heart, Lung and Blood Institute. The DSMB includes critical care physicians with phase 1/2 trial experience and a biostatistician. The DSMB is required to provide recommendations about starting, continuing, and stopping the study. In addition, the DSMB is asked to make recommendations, as appropriate, to the NHLBI about: benefit/risk ratio of procedures and participant burden; selection, recruitment, and retention of participants; adherence to protocol requirements; completeness, quality, and analysis of measurements; amendments to the study protocol and consent forms; performance of individual centers and core labs; participant safety; notification of and referral for adverse events.Since this is a phase 1/2 clinical trial focused on safety and since the patient population is critically ill and at high risk for complications related to their underlying severity of illness, we have also appointed a designated external medical monitor and constituted a scientific review committee, chaired by the external medical monitor. In phase 1, the scientific review committee plays a critical role in evaluation of each patient in each dosing cohort to ensure patient safety. Specifically, in phase 1, the first subject in each cohort who receives the MSC infusion is observed for seven days prior to enrollment of the remaining subjects in that cohort. A report of clinical data and adverse events from the first subject in the cohort is reviewed by the scientific review committee. If the patient meets stopping criteria (pre-specified infusion associated event or serious adverse event including death within seven days) or there is concern on the part of the scientific review committee, enrollment will be suspended pending review by the DSMB. If stopping criteria are not met, and there are no safety concerns, the next two subjects in that cohort may be enrolled. The second and third subjects in the cohort may be enrolled concurrently; however if the second subject experiences a pre-specified clinically important event or unexpected serious adverse event, including death, prior to enrollment/dosing of the third study subject, enrollment/dosing will be suspended pending review by the DSMB. After completion of enrollment of each study cohort and seven days of follow-up for all three individuals in the cohort, an aggregate report of clinical data and all adverse events will be reviewed by the scientific review committee and DSMB for each cohort. After completion of the phase 1 study , the scientific review committee will review the data and propose a cell product dose for the phase 2 study. This recommendation will be submitted to the DSMB for approval prior to initiating the phase 2 study.In phase 2, the external medical monitor will assist the study with adjudication of severe adverse events on a case-by-case basis.Phase 1/2 trials of a cell-based therapy in critically ill subjects pose unique design challenges, including the need for clearly defined criteria for clinical stability prior to study treatment and the need to define infusion associated adverse events in order to try to separate medical events related to the patient’s overall condition from events that could be related to the cell-based therapy. The need to define ‘baseline stability’ poses additional challenges related to the need to coordinate cell administration with the bone marrow transplantation laboratory. We describe here some of our proposed solutions to these issues, which should be applicable to both cell-based therapy trials for other diseases, as well as most other early phase trials based in the ICU.ARDS: Acute respiratory distress syndrome(APACHE) III: Acute Physiology, Age, Chronic Health EvaluationDSMB: Data safety monitoring boardLIS: Lung injury scoreMTD: Maximally tolerated doseMSC: Mesenchymal stem cellsOI: Oxygenation indexPEEP: Positive end-expiratory pressureSOFA: Sequential Organ Failure AssessmentNone of the authors has any competing  interests to declare.KDL and MAM conceived of the study, and participated in its design and coordination and helped to draft the manuscript. HZ was responsible for study coordination throughout the design and implementation phases. LC, MM, DM and AL were responsible for study design and implementation relevant to MSC harvest and well as processing in the bone marrow transplantation lab. XF was responsible for the design and implementation of potency assays for the trial. KC assisted with design and rollout of the study at subsites. JWL was involved in the design of the cell wash protocol. JGW, CSC, JG, AR, JL, KNK, JW-K and BTT were involved in study design. KLD assisted with the statistical analysis plan. All authors read and approved the final manuscript.


Consultant surgical practice including the outcomes has been under great scrutiny from day 1. Having a solid foundation in the early stages is going to give confidence to take high risk cases in the later years with good results. With this I am going to present my own series of first 100 cases.Here I would like to present, how I performed first 100 operations as a consultant surgeon without mortality, stroke, re-exploration, deep sternal wound infections or mediastinits. I take this opportunity to share my methodology of case selection and preparation for surgery with the upcoming consultant colleagues and senior trainees who are going to be the future generation of cardiac surgeons.This is prospective, which was retrospectively collected on my patients whom I operated between July 2010 and March 2011 as a new consultant. The total 105 cases includes 76 CABG, 21AVR, 8 AVR+CABG and third of these were acute cases. The mean age was 59.3 (5 - 84) and mean additive Euro score of 4.5 (0-10) and only 10% are mod -poor LV function. None of the patients had IABP either before or after surgery.There were no post-op mortality or stroke and none of the patients were re-explored for bleeding or tamponade. Only 2 patients were readmitted to ICU for tachyarrythmias and 3 patients needed re-intubations (out of which one needed tracheostomy). 10% had minor postop complications including small left sided pleural effusions, pneumothorax and superficial leg wound infections. None of these patients had deep sternal or leg wound infections requiring vac therapy or rewiring.Careful patient selection and attention to detail are the key factors for achieving good postoperative outcomes. Career as a consultant cardiac surgeon is a challenging journey. We are practising in times where outcomes are highly scrutinised and available to public domain. Gone those days where people are going to accept mishaps (just because you started as a newly appointed consultant). Good foundation in the initial stages gives you the confidence to handle challenging and high risk cases in the future.


The composition of cell walls in cereal grains and other grass species differs markedly from walls in seeds of other plants. In the maternal tissues that surround the embryo and endosperm of the grain, walls contain higher levels of cellulose and in many cases are heavily lignified. This may be contrasted with walls of the endosperm, where the amount of cellulose is relatively low, and the walls are generally not lignified. The low cellulose and lignin contents are possible because the walls of the endosperm perform no load-bearing function in the mature grain and indeed the low levels of these relatively intractable wall components are necessary because they allow rapid degradation of the walls following germination of the grain. The major non-cellulosic components of endosperm walls are usually heteroxylans and -β-glucans, with lower levels of xyloglucans, glucomannans, and pectic polysaccharides. Pectic polysaccharides and xyloglucans are the major non-cellulosic wall constituents in most dicot species, in which -β-glucans are usually absent and heteroxylans are found at relatively low levels. Thus, the “core” non-cellulosic wall polysaccharides in grain of the cereals and other grasses are the heteroxylans and, more specifically, arabinoxylans. The -β-glucans appear in the endosperm of some grass species but are essentially absent from others; they may constitute from zero to more than 45% of the cell walls of the endosperm, depending on the species. It is clear that in some cases these -β-glucans function as a major store of metabolizable glucose in the grain. Cereal grains and their constituent cell wall polysaccharides are centrally important as a source of dietary fiber in human societies and breeders have started to select for high levels of non-cellulosic wall polysaccharides in grain. To meet end-user requirements, it is important that we understand cell wall biology in the grain both during development and following germination. Two major differences distinguish the cell walls of cereal grains from those found in seeds of other higher plant species. Firstly, the cell walls of the Poaceae family, which includes the grasses as well as the economically important cereals, are fundamentally different in composition, compared with walls in dicotyledons and in most other monocotyledons. Secondly, walls in the grain of the Poaceae are usually quite different than those found in vegetative tissues. Here we will examine emerging evolutionary evidence and potential selection pressures that might account for these two levels of differences in wall composition in cereal grains.in situ methods to define the heterogeneity of wall composition in plant material that contains different cell types. Thus, sophisticated methods for determining polysaccharides present in walls during grain development are under development. For example, there has been a recent surge in the availability of reliable antibodies and carbohydrate binding modules that detect specific epitopes on wall polysaccharides  and a range of cell shapes and sizes across the grain. In barley there are wings of irregularly shaped starchy endosperm cells that flank a central core of prismatic cells overlying the transfer cells  and sorghum and maize grain can be dominated by one particular type of endosperm and thus can be predominantly soft or hard . In both cellularizing barley and rice endosperm callose is believed to be the major component of the cell walls that grow around the nuclei in the syncytium. In barley callose is found along the central cell wall at 3 days after pollination (DAP); it is present in the first and subsequent anticlinal walls from 4 DAP, in the periclinal walls at 5 DAP and disappears at 6 DAP, except in the vicinity of plasmodesmata  or more of primary walls in vegetative tissues . The lowMost of the discussion above has been focused on the development of cell walls in the starchy endosperm of grains of the Poaceae. However, as the grain develops several other specific cell types can be distinguished. These include, in addition to the starchy endosperm cells, which are the main repository for starch and storage protein, TC, which are clustered around the vascular network that feeds the growing grain, aleurone cells, which envelop the starchy endosperm and are rich in oil and protein bodies, sub-aleurone cells that arise through periclinal division of the aleurone cells, and finally the embryo itself, which is comprised of many organ-specific vegetative tissues. Information on the cell walls of these tissues is not extensive, but some interesting data are emerging.Aleurone cells form a layer around the starchy endosperm that varies from one to three or four cells in thickness, depending on the species, and are indeed components of the endosperm as a whole. Aleurone cells are typically cuboid in shape with much thicker cell walls, usually at least twice the thickness of those in the central starchy endosperm. Aleurone cells contain a dense granular cytoplasm comprised of aleurone grains and small vacuoles containing inclusion bodies . They arThe walls of aleurone cells in mature barley and wheat grain have two quite distinct layers . The innThe developmental cues for aleurone cells are complex and not yet fully understood. In wheat, they have a specific molecular signature by 6 days post anthesis, conferred by their position in the “surface layer” . HoweverB. distachyon and Festuca pratensis have markedly more disorganized and irregular aleurone layers, which may imply that there is a correlation between regularity of shape and domestication, since this trait may have been selected to meet certain grain quality parameters, such as speed of germination and endosperm mobilization -β-glucan are found in wheat and barley grain. Early work in which aleurone cells were isolated and analyzed showed that aleurone walls from wheat and barley contained about 65% arabinoxylan and about 28% -β-glucan; cellulose and glucomannan levels were again very low . Severalmaturity . Antibodmaturity .p-coumaric acid in mature aleurone walls in wheat . Reticulate TCs arise from re-differentiation of epidermal cells -β-glucans, while cellulose contents are usually low, as noted above . HoweverAlthough arabinoxylan and -β-glucan predominate in cereal grain cell walls, we are starting to discover the presence of other polysaccharides which, although only minor components of the walls, may represent key determinants of wall plasticity and other properties. Thus, levels of pectic polysaccharides, heteromannans, and xyloglucans are low in many grains, including wheat and barley . Again aB. distachyon -β-xylan backbone with arabinofuranosyl residues sterically hinders the aggregation of the -β-xylan chains into insoluble microfibrils and results in the formation of a long, asymmetrical polysaccharide that is partly soluble in water and can form gel-like structures in the cell wall matrix . As expeEquisetum spp. horsetail ferns -β-glucan. This polysaccharide has an interesting distribution in the plant kingdom . It is fil ferns , bryophyil ferns , some fuil ferns , brown, il ferns , and licil ferns . This diThe -β-glucans of the grasses are comprised of an unsubstituted chain of glucosyl residues linked either through -β- or -β-linkages. About 90% of the polysaccharide chain is comprised of cellotriosyl (DP3) and cellotetraosyl (DP4) units that are linked through -β-linkages; adjacent β-linkages are rare or absent . ApproxiTable 1; B. distachyon, where this polysaccharide has a ratio of 5.8:1 and clearly has evolved to perform a storage function , their physical properties are similar and well adapted to a structural role in cell walls. This is therefore an example of convergent evolution to the extant state. Arabinoxylans are extended asymmetrical molecules by virtue of their linear -β-xylan backbone and are partly soluble because of the steric hindrance of intermolecular aggregation afforded by their arabinofuranosyl substituents. Solubility is further influenced by acetylation and feruloylation which participate in cross-link formation between arabinoxylan and other wall components. This is exemplified in wheat endosperm walls where the degree of acetylation declines affecting solubility as the grain matures -β-glucans are quite different -β-glucans with lichenase and reported that the DP3:DP4 ratio was elevated to 7:1 in younger endosperm, compared with around 4:1 in mature tissue.-β-Glucan is the predominant polysaccharide in the starchy endosperm cell walls of barley and oats and comprises about 15% of starchy endosperm cell walls in wheat grain . RecentlCesA clade encodes cellulose synthases (Csl) clades of the gene superfamily have been implicated in the synthesis of different wall polysaccharides. The CslA group of genes is likely to encode mannan and glucomannan synthases -β-glucan backbone of xyloglucans and the genes in the CslD clade may be involved in cellulose synthesis, particular in cells that exhibit tip growth -α-L-arabinosyl transferases -α-Araf-(1 → 3) substitutions  contain xylan that is almost completely unsubstituted, but still contain wild-type amounts of the xylan backbone. This indicates that the synthesis of the backbone and its substitution can be uncoupled; a somewhat surprising observation when the behavior of such an unsubstituted and hence possibly insoluble polysaccharide in an aqueous environment is considered, although potential insolubility may be ameliorated by extensive acetylation. The domain of unknown function protein, DUF579, which was reported by O-methyl transferase that catalyzes the methyl etherification of C(O)4 of glucuronyl residues in heteroxylans of Arabidopsis -β-glucans. In the case of the arabinoxylan enzymes, much of the initial work on the identification of genes involved was focused on analyses of families . Howeversferases , whilst itutions . Zeng et8 family . Double bidopsis .CslF and CslH clades of the cellulose synthase gene superfamily. These genes are found only in the Poaceae -β-glucans in the walls of transgenic plants -β-glucans in its walls and does not have CslF or CslH genes. These genes are members of smaller gene sub-families that contain about 10 CslF genes and just a few CslH genes -β-glucan content in the transgenic barley grain -β-glucan in its grain -β-glucan synthesis and to investigate this possibility genome-wide association mapping has been used in attempts to identify other genes that might contribute to the biosynthesis or regulation of -β-glucan synthesis -β-glucans are reasonably well defined and include members of the  Poaceae  and whenc plants . As a dilH genes . It has ey grain . Similarts grain . It is wynthesis .CslF and CslH clades evolved from other clades in the cellulose synthase gene superfamily. The CslF and CslH clades are not particularly close on the phylogenetic tree -β-glucan synthases is not known. However, it is clear that some competitive advantage must be associated with the presence of -β-glucans in walls of the Poaceae and that selection pressure has retained the capacity of enzymes encoded by CslF and CslH genes to synthesize -β-glucans. Detailed phylogenetic analyses indicate that the CslF genes shared a common ancestor with CslD genes and are now under a stationary selection barrier . In the Bd21 line there is a concomitant drop in grain starch content from values of 60–65% that are typical for grains of the Triticeae to 6% w/w -β-glucan synthase genes, notably BdCslF6, were not affected. This lead to the hypothesis that the thick walls in B. distachyon grain are the result of continued accretion of -β-glucan onto walls of cells that are not expanding -β-glucan that they contain; this can vary from close to zero in rice to 45% w/w in the starchy endosperm of yon Bd21 . The stao 6% w/w . Trafforxpanding . Even thB. distachyon uses -β-glucans as a storage polysaccharide in its grain, where small amounts of starch are present ?The reasons for the variability of -β-glucan content in cereal grains is not known, but it has been suggested that this polysaccharide acts as a secondary store of metabolizable glucose and that this function might be the key to the adoption of -β-glucans during the evolution of the grasses . It is c present . There i present . During  present . It has  present . Might tB. distachyon storage metabolism seems to have shifted to -β-glucan for unknown reasons, although disadvantageous mutations in starch synthase and cell cycle genes are possible explanations -β-glucan.Where an alternative storage carbohydrate is found in storage tissues, one would expect to see a battery of corresponding hydrolytic enzymes expressed in the germinated grain or seed, to catalyze the efficient breakdown of the polysaccharide into component sugars for use by the growing embryo and young seedling. There is little information available on germination processes in The distinguishing features of cell walls in the grasses include the adoption of heteroxylans as the “core” non-cellulosic polysaccharide and the corresponding lower levels of xyloglucans and pectic polysaccharides. The widespread adoption of -β-glucans in the Poaceae family also distinguishes the grasses from other monocotyledonous and dicotyledonous plants, although it is intriguing that -β-glucans do not appear to be an essential structural component of walls in these species. One might question whether these distinguishing characteristics of cell walls of the grasses and their grain might in any way contribute to the obvious evolutionary success of the Poaceae family. It has been estimated that the grasses, which have appeared relatively recently in evolutionary history , now domHvCslF6 gene in barley led to more than 50% increases in dietary fiber in the transgenic grain (in situ methods will greatly facilitate that research.The non-cellulosic wall polysaccharides of plants are important components of dietary fiber and increased intake of dietary fiber has been advocated to reduce the risk of contracting serious human diseases, including colorectal cancer, type II diabetes, and cardiovascular disease . Over-exic grain . Given tThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dyslexia is characterized by slow, inaccurate reading. Previous studies have shown that the Reading Acceleration Program (RAP) improves reading speed and accuracy in children and adults with dyslexia and in typical readers across different orthographies. However, the effect of the RAP on the neural circuitry of reading has not been established. In the current study, we examined the effect of the RAP training on regions of interest in the neural circuitry for reading using a lexical decision task during fMRI in children with reading difficulties and typical readers.Children (8–12 years old) with reading difficulties and typical readers were studied before and after 4 weeks of training with the RAP in both groups.In addition to improvements in oral and silent contextual reading speed, training-related gains were associated with increased activation of the left hemisphere in both children with reading difficulties and typical readers. However, only children with reading difficulties showed improvements in reading comprehension, which were associated with significant increases in right frontal lobe activation.Our results demonstrate differential effects of the RAP on neural circuits supporting reading in both children with reading difficulties and typical readers and suggest that the intervention may stimulate use of typical neural circuits for reading and engage compensatory pathways to support reading in the developing brain of children with reading difficulties. Developmental reading disability (RD) or dyslexia is characterized by slow, inaccurate reading that cannot be attributed to sensory difficulties, low IQ, or educational deprivation. Reading difficulties typically continue into adulthood despite remedial intervention and repeated exposure to the written language Fletcher . IndividThe main normalization strategy reported in the literature for individuals with RD and the goal of most interventions for this population, focus on a shift in neural activation from the right brain hemisphere to the left. At baseline, several studies have reported an atypical increase in activation of the right hemisphere during reading, as measured by either functional MRI (fMRI) . Alternatively, with intensive reading training these children might begin to engage more typical neural circuitry to support improved reading skills . A third possibility, one that we hypothesize is most likely to occur in the developing brain of a child who is struggling to learn to read, is a mixture of the two strategies in which domain-specific reading intervention in children with RD would increase engagement of typical reading circuits while also enhancing the efficiency of compensatory pathways. In this study, we will test this hypothesis using fMRI, behavioral testing of reading performance, and a computer-based reading intervention program known as the Reading Acceleration Program (or RAP).The RAP  to examine remodeling of the neural substrates for reading in children with RD after 4 weeks of training with the RAP. Similar imaging and training is performed in TRs to allow us to demonstrate that the influence of training with the RAP is specific in children with RD. Behavioral reading measures are used to assess the change in reading ability following RAP training in both groups of children. Based on the theory of how the RAP works to improve reading, we can make several predictions about what effect the training will have on reading performance and brain activity patterns in children with RD and TRs. First, we predicted that (1) both children with RD and TRs who train on RAP would show improved reading speed, accuracy, and comprehension as a result of training with the RAP, as demonstrated previously  and wait-list groups (n = 15 children with RD). Members of the experimental group received the RAP training intervention, whereas the wait-list group has not received the training but had an opportunity to use the RAP upon completion of the study. All participants were native English speakers, right-handed, displayed normal or corrected-to-normal vision in both eyes, and had normal hearing. None had a history of neurological or emotional disorders. Children with RD and TRs were recruited from posted ads and through commercial advertisement. Participants in the TRs group were students of the same chronological age who volunteered for the study and had fluent and accurate reading (according to accepted norms).Thirty-three children with RD  and 18 TRs  participated in the current study. Nonverbal IQ scores were determined for all participants using the Test of Nonverbal Intelligence – 3rd edition (TONI-3) , but were not scanned. After completion of the second assessment, wait-list participants were invited to participate in the RAP training.All participants gave their informed written assent and their parents provided informed consents prior to inclusion in the study, and all were compensated for their participation. The Cincinnati Children's Hospital Medical Center (CCHMC) Institutional Review Board approved the experiment. The study was carried out in the imaging center of the CCHMC Pediatric Neuroimaging Research Consortium (PNRC) in Cincinnati, Ohio.Children with RD either received previous diagnoses or parents had reported their children as having reading difficulty (confirmed by the study's reading battery). Reading ability in both children with RD and TRs was evaluated using a battery of normative reading tests in English: (1) untimed single-word reading ability . Both children with RD and TRs in the experimental group underwent baseline behavioral and neuroimaging assessment, 4 weeks of training with the RAP, and follow-up behavioral and neuroimaging assessments.Attention was assessed using Conners' Rating Scales – Parent Rating Scales and Self-Report Scales Conners . These mReading assessment section) were determined (Test 1). The reading measures were used to assess the effects of the RAP  as well as silent reading speed and comprehension measures from the RAP evaluation mode. Reading measures were also administered to the wait-list group to eliminate the effects of motivation or exposure to the reading tests on reading scores in children with RD in the experimental group. Each reading assessment lasted approximately 1.5 h.In order to enroll the participants into one of the two reading groups (children with RD vs. TRs), baseline word reading, decoding, and fluency measures .t-tests were performed in order to reveal the source of the interaction.To assess the main effects of Group, Test, and the Group × RAP training interaction following the RAP training on different reading levels , we performed separate repeated measures analyses of variance (RM ANOVAs) on each of the reading measures. Post hoc paired and independent Since the RAP trains contextual silent reading and the fMRI task employs a single-word recognition paradigm, we correlated the contextual reading scores  with word reading from the TOWRE-II battery. A Pearson correlation was performed for the entire sample.Both children with RD and TRs in the experimental group (those receiving the RAP training) completed two MRI sessions both before and after the reading training that included alignment and anatomical scans followed by an fMRI paradigm. The effect of the RAP intervention is related to orthographic patterns  was used for presentation of the stimuli as well as a movie during the preparation  and acquisition of the whole-brain anatomical scans. A gradient echo planar sequence was used for T2*-weighted BOLD fMRI scans with the following parameters: TR/TE = 2000/38 msec; BW = 125 kHz; FOV = 25.6 × 25.6 cm; matrix = 64 × 64; slice thickness = 5 mm. Thirty-five acquired slices covered the entire cerebrum. Seventy-eight image volumes were acquired during the fMRI experiment consisting of 30 sec per condition for a total acquisition time of 2 min and 36 sec. A 3D T1-weighted inversion recovery gradient echo anatomical whole-brain scan also was acquired from each participant for anatomical coregistration and for use in spatial normalization of the functional MRI data.http://www.fil.ion.ucl.ac.uk/spm/), images were slice-time corrected and realigned. Data were normalized using the 3D anatomical whole-brain scan (7th degree-spline interpolation) to match the Montreal Neurological Institute (MNI) standard template, resampled (3 mm3 voxels), and smoothed with 8-mm full width at half maximum (FWHM). A general linear model approach was used to identify voxels activated by the task for each participant. The second level analysis was based on the individual contrast images (words > pseudowords).Using SPM8  that were previously reported to show activation during word reading. Ten 10) of these regions were derived from meta-analyses of reading . To test the effect of training in the two reading groups, we performed a two-way ANOVA, treating Group as a two-level factor (children with RD and TRs) as well as Training with the RAP . The ANOVA and pairwise comparisons were performed on the beta values of the main effect variable  for the contras of words > pseudowords in each region, with a small-volume correction for multiple comparisons. This approach takes into account all of the voxels in the ROI and the small-volume correction is done across those.For each ROI, group-level analyses were carried out using a random effects ordinary least-squares model. Small-volume correction  for multiple comparisons was performed using Gaussian Random Field Theory  or that showed a significant Group × Training with the RAP interaction, were interrogated by post hoc analysis using independent In the same set of ROIs, we used multiple regressions to explore relationships between the activation in each region after training with the RAP and the magnitude of gains in reading performance . Since the RAP improves reading speed, accuracy, and fluency (Horowitz-Kraus and Breznitz http://www.wordfrequency.info/). Each stimulus is a sentence with a multiple-choice question followed by four possible answers. Each sentence length is 9–12 words, comprised 45–70 letters, letter width of 5 mm, extending over 1 to 2 lines and with 18 mm between lines. Each sentence is presented once during the entire training intervention. The RAP sentences have been tested and verified for their level of difficulty in previous studies . In the first training session, 50 sentences were presented consecutively on the screen. The letters in each sentence disappeared one after the other, according to the mean reading time (msec per letter) recorded on the pretest. Following the disappearance of the sentence from the computer screen, participants were instructed to answer a comprehension question. The per-letter “presentation rate” decreased from one sentence to the next in by 2% for each subsequent sentence Breznitz ,b and tht-tests between participants in the RDs and TRs groups, and between the wait-list and experimental (trained) RDs groups at baseline (Test 1) revealed no significant differences in IQ or attention measures  RM-ANOVAs:1. Effect of the RAP on silent reading speed and comprehension .Speed: Main effects of Training with the RAP  and Group  indicating faster reading speed after training with the RAP and a generally slower reading pace in children with RD compared to TRs.Comprehension: Main effects of the Training with the RAP  and Group  indicating greater comprehension after training with the RAP and lower reading comprehension scores in children with RD compared to TRs. The significant Group × Training with the RAP interaction  indicates a greater change following training in comprehension scores in children with RD as compared to the TRs .Speed: Main effects of Training with the RAP  and Group  indicating faster reading speed after the RAP training and a generally slower reading pace in children with RD compared to TRs.Comprehension: Main effects of Training with the RAP  and Group  indicating greater comprehension after training with the RAP in both groups and generally lower comprehension in children with RD (see Table 3. Effect of the RAP training on word and pseudoword reading (data derived from the TOWRE-II).Word reading: Main effects of Training with the RAP  and Group  indicating greater reading scores after training with the RAP and lower word-reading scores in children with RD compared to TRs.Pseudoword reading: Main effects of Training with the RAP  and Group  indicating greater pseudoword reading scores after training with the RAP in both groups and generally lower scores in children with RD compared to TRs , accuracy , and comprehension . Our results suggest that greater word-reading ability is associated with reading comprehension and more accurate, faster contextual oral reading. This is consistent with previous results highlighting the correlation of these skills in children  with the scores of the word reading task from TOWRE-II. A Pearson correlation was performed for the entire sample. The analysis revealed that word-reading scores were positively correlated with contextual reading rate (GORT-IV)  for children with RD and TRs for Tests 1 and 2 were performed. Results demonstrate that before intervention  the left PCG, STG, IOG, and right IPL were activated in TRs, and after intervention only the left PCG was significantly activated (Test 2- red dots)  in the left and right inferior occipital gyrus , left inferior frontal gyrus triangularis , and right precentral gyrus . A significant main effect of Training with the RAP  was found in the left occipital gyrus , left superior temporal gyrus , and left medial frontal gyrus . A significant Group × Training with the RAP interaction  also was found in the right anterior cingulate cortex , right medial frontal gyrus , right inferior frontal gyrus opercularis (rIFGop BA 44), and right inferior frontal gyrus triangularis . No activation was found for pseudowords > words contrast.A two-way ANOVA, with Group (two levels: Children with RD and TRs) and Training with RAP (two levels: Test 1 and Test 2) for each chosen ROI was performed using small-volume correction  of regions exhibiting a main effect of Group, Training with the RAP, and Group × Training with the RAP interaction revealed that the main effect of Group resulted from the greater activation in children with RD in the lIOG and rIOG (BA 18), rPCG (BA 4), and lIFGtr (BA 46). The main effect of Training with the RAP was attributed to greater activation of the lIOG (BA 18) and the lSTG (BA 41) after intervention (Test 2) and the lMFG (BA 9) before intervention (Test 1). The interaction resulted from a greater activation in the rACC (BA 32) in children with RD in Test 2 versus Test 1, greater activation in the rIFGop (BA 44) and IFGtr (BA 46) in children with RD than TRs in Test 2, and greater activation in the rMFG (BA 9) in children with RD than TRs in Test 1.Post hoc analysis  across all voxels in each ROI from Test 2 with the gain in reading scores (the difference between Test 1 and Test 2) for contextual reading rate, contextual reading accuracy (GORT-IV), and word/pseudowords fluent reading (TOWRE-II). After small-volume correction, the following significant correlations were found Figs and 4:Children with RD: Children with RD showed significant positive correlations between the gain in scores for contextual reading rate (GORT-IV) and the activation of the lACC (BA 32), and between the scores for the gain in contextual reading accuracy (GORT-IV) and the activation of the lMFG (BA 9). Significant positive correlations also were found between word/pseudoword efficiency reading scores and the activation of the lMFG (BA 9), lFFG (BA 37), and lIOG (BA 18) .Typical readers: TRs showed significant positive correlations between gain in scores for contextual reading rate (GORT-IV) and the activation of the rMFG (BA 9) and rIPL (BA 40), and between the scores for the gain in contextual reading accuracy (GORT-IV) and the activation of the rIFGtr (BA 46) and rIPL (BA 40). Significant positive correlations also were found between word/pseudoword efficiency reading scores and the activation of the rIPL (BA 40) .No significant correlations between the levels of activation in the ROIs in Test 2 and rate/comprehension measures from the RAP were found.The current study aimed to discover whether children with RD in the experimental group showed reorganization in neural pathways related to reading making them similar to TRs  or alternative neural patterns that differed from TRs . Another aim of our study was to explore the effect of the RAP training on neural circuits underlying reading in TRs.Our results indicate that both children with RD and TRs exhibited improvements in oral and silent reading following 4 weeks of training with the RAP, which is consistent with our hypothesis. We found that children with RD demonstrated faster oral and silent reading, greater comprehension scores as well as more accurate reading following intervention. These findings were not observed in our wait-list group of children with RD, allowing us to infer that the effect was due to the RAP training rather than a placebo effect associated with enrolling in a research protocol. These results confirm previous findings of the positive effect of the RAP in children with RD  and frontal  activation than TRs. The absence of left hemisphere specialization in reading and particularly the lack of focal activation in the occipital regions may represent the neural correlates of dyslexia , which shifted primarily to the left after the training . TRs also demonstrated a greater activation in the left PCG after training. The activation of the regions related to orthographical processing  were previously found to be more active during exposure to words  in both children and adults , children with RD still showed activation in the right hemisphere during reading, especially in the rIPL. Surprisingly, TRs also showed positive association between the activation of the rIPL and greater reading gain after training with the RAP. The rIPL is primarily involved in phonological processes  following training observed in both children with RD and TRs. This activation might be related to the postulated effect of the RAP on working-memory abilities  activation after training with the RAP in children with RD may be due to greater error monitoring  together with more basic higher order abilities such as executive functions  . Previous studies have shown significant behavioral changes in reading performance after 8 weeks of one-on-one training , to keep our focus on the contrast relevant to executive functions associated with reading. This limited the data stream in our analysis to 156 sec consisting of only 78 image volumes. Despite the limited signal-to-noise ratio in the resulting contrast maps, we found statistically significant ROI-based differences between groups and sessions that survived Bonferroni correction for multiple comparisons. Whole-brain exploratory analysis of differences in activation, either between groups or before and after training, was not possible in this case, since differences likely would not survive corrections for multiple comparisons at the voxel level. Consequently, we limited our analysis to brain regions that we a priori hypothesized would be influenced by the RAP training. These hypotheses were based on prior work with the RAP and other imaging studies in children with RD. Hypothesis-driven ROI analyses produced significant results that allowed us to explore hypotheses about neuroplastic changes in the neural circuitry of reading that correspond to normalization versus compensation strategies in the developing brain and that fit well into the context of prior literature regarding interventions for individuals with RD. Whole-brain analyses will require a future study with a larger number of subjects and longer duration neuroimaging paradigms. However, even with the short duration of training and the limited neuroimaging assessments, we have found evidence of interactions of neuroplasticity and reading improvements in children with RD in response to the RAP, providing ample evidence for further investigations into the mechanisms by which the RAP improves reading performance using DTI, fMRI, and morphometric analysis of anatomical brain images.Third, the current study demonstrated that the effect of training with the RAP is specific to the trained group through a lack of change in reading measures in the wait-list group based on behavioral reading measures. We did note, however, that although the wait-list group did not show a significant increase in reading scores, we still did not observe a significant change between the trained or wait-list of children with RD  may respond differently than other children with RD and TRs to the same reading programs. Also, the greater activation of frontal regions  in children with RD after training with the RAP supports previous findings of executive function improvement following this training (Horowitz-Kraus and Breznitz


Understanding post-launch demand for new vaccines can help countries maximize the benefits of immunization programmes. In particular, low- and middle-income countries (LMICs) should ensure adequate resource planning with regards to stock consumption and service delivery for new vaccines, whereas global suppliers must produce enough vaccines to meet demand. If a country underestimates the number of children seeking vaccination, a stock-out of commodities will create missed opportunities for saving lives. We describe the post-launch demand for the first dose of pneumococcal conjugate vaccine (PCV1) in Ethiopia and Malawi and the first dose of rotavirus vaccine (Rota1) in Malawi, with focus on the new birth cohort and the ‘backlog cohort’, comprised of older children who are still eligible for vaccination at the time of launch. PCV1 and Rota1 uptake were compared with the demand for the first dose of pentavalent vaccine (Penta1), a routine immunization that targets the same age group and immunization schedule. In the first year, the total demand for PCV1 was 37% greater than that of Penta1 in Ethiopia and 59% greater in Malawi. In the first 6 months, the demand of Rota1 was only 5.9% greater than Penta1 demand in Malawi. Over the first three post-introduction months, 70.7% of PCV1 demand in Ethiopia and 71.5% of demand in Malawi came from children in the backlog cohort, whereas only 28.0% of Rota1 demand in Malawi was from the backlog cohort. The composition of demand was impacted by time elapsed since vaccine introduction and age restrictions. Evidence suggests that countries’ plans should account for the impact of backlog demand, especially in the first 3 months post-introduction. LMICs should request for higher stock volumes when compared with routine needs, plan social mobilization activities to reach the backlog cohort and allocate human resources and cold chain capacity to accommodate high demand following vaccine introduction. Key MessagesUnderstanding post-launch demand for new vaccines can help countries maximize the benefits of immunization programmes. When planning a new vaccine introduction, countries should consider the impact of the ‘backlog cohort’ of age-eligible children born before the introduction of the vaccine, especially in the first 3 months of introduction.In the case of Ethiopia and Malawi, in the first year of pneumococcal conjugate vaccine (PCV) introduction, the demand for the new vaccine (PCV1) was 37% and 59%, respectively, greater than that of the benchmark vaccine (Penta 1), mostly due to demand from older ‘backlog’ children.Countries can maximize the life-saving potential of immunization through vaccinating a larger share of the ‘backlog cohort’ by setting looser age eligibility criteria.To cater to this backlog cohort, countries’ plans should request high stock volumes for the new vaccines, allocate adequate human resources and cold chain capacity to accommodate backlog cohort demand, and include social mobilization activities to reach more children in the backlog cohort and reduce drop-out.A significant wave of new vaccine introductions (NVIs) is taking place worldwide. For instance, 124 countries have already introduced the pneumococcal conjugate vaccine (PCV), whereas 25 countries are planning its introduction . Seventyhow to best introduce these new vaccines.Although recent immunization research has focused on analysing factors leading to the decision to introduce a new vaccine  every 5 years, Ministries of Health and their partners complete a situational analysis of the national epidemiological context and immunization system. From this assessment, countries identify the new vaccines that should be included in their national programme, and define tentative dates for introduction as part of the multi-year workplan. Currently Gavi-eligible countries submit an expression of interest, followed by an application for support to Gavi, the Vaccine Alliance. Indeed, availability of Gavi funding is one of the main determinants of countries’ deciding to introduce a vaccine . If the One difficult decision at the time of the Gavi application is the forecast of demand for the vaccine: countries must estimate how many children will receive the first and last dose of the vaccine during each year of the programme for the cMYP period . CharactToday, supply forecasting exercises for NVI typically estimate future consumption based on the number of age-eligible children born in the year after introduction (i.e. the new birth cohort) as the demand, and factor in wastage and a buffer stock to estimate the number of doses received in Year 1. However, the actual demand for vaccines may also include a ‘backlog cohort’, the older children who are eligible for vaccination at the time of introduction. For instance, the routine immunization schedules in Ethiopia and in Malawi stipulate that children be vaccinated with the first dose of PCV at 6 weeks of age, but children remain eligible for this vaccine until their first birthday. Thus, children younger than 6 weeks at the time of introduction and those born in the year after introduction are part of the ‘new birth cohort’. But children between 6 weeks and 1 year of age at the time of introduction may also seek vaccination, and these children constitute the backlog cohort.Neither Gavi guidelines nor the cMYP costing tool guidance  refer toWhere age eligibility rules have been chosen based on a careful understanding of medical benefits, countries can generally maximize the life-saving potential of immunization by vaccinating a larger share of the backlog cohort. This is true if vaccination elicits seroconversion—and thus protection against disease—in older children within the backlog cohort before natural infection occurs. In addition, the risk of disease acquisition must continue after the date of the last scheduled dose, as was the case in Ethiopia and Malawi. Indeed, 31% of all hospitalized WHO clinical pneumonia cases in Africa occurred in children aged 12–23 months . LimitinOnce age eligibility rules are set, failure to incorporate the backlog cohort demand into NVI planning can lead to suboptimal outcomes. For instance, if a country underestimates the consumption of a new vaccine by the backlog cohort, the introduction of the new vaccine will yield partially vaccinated or unvaccinated children due to stock-outs or rationing of the new vaccines.There is a paucity of evidence on the quantitative composition of demand between new and backlog cohorts. In particular, no published articles have until now conducted analysis of health facility-level child vaccination records to understand uptake dynamics over the first year of a vaccine introduction. As a result, little guidance exists on the backlog cohort’s impact on stock and routine immunization administration in the introduction year.This study offers a novel empirical comparison of three vaccine introductions. We are able to compare the introduction of two different vaccines (three-dose PCV13 and two-dose Rota) in Malawi, and compare the introduction of the same vaccine (three-dose PCV13 and three-dose PCV10) in two different countries . The Clinton Health Access Initiative (CHAI) collaborated with Ministries of Health in Ethiopia and in Malawi on this study to inform the national vaccine programmes in each country and to ensure the reach of the new vaccines was quickly maximized. Beyond Ethiopia and Malawi, our findings provide critical evidence for practitioners worldwide planning NVIs and for their suppliers on how the composition of demand can impact launch dynamics including forecasting, stock management, service delivery and drop-out rates.The study population included children receiving publicly available new and routine immunizations in Ethiopia and Malawi in 2011–13. Uptake of new vaccines was compared with pentavalent vaccine (Penta), which was already established in the routine immunization system and targets the same age group and immunization schedule.Health facility-based data were collected in Ethiopia on three doses of PCV , and three doses of Penta  by obtaining vaccination records of all children after PCV launch (October 2011). Data were extracted for all children who were registered in the health facility child immunization registration book as vaccinated within 1 year after PCV launch. Records in Ethiopia included an individual identifier that allowed for tracking vaccinations received for a single child over time.In Malawi, data were extracted from health facility registers on the same vaccines as well as both doses of Rota (Rota1 and Rota2) for two time periods: 1 year post-PCV launch (November 2011–October 2012) and then again for 6 months post-Rota launch (October 2012–March 2013). Each immunization is recorded on one line of the facility register with date of vaccination and the birthdate of the receiving child. These records were captured for all days within the two time periods. It was not possible in Malawi to track vaccinations received by a single child over time, as this is not recorded. Our data are therefore cross-sectional in nature.To ensure data were collected from a representative sample of health facilities, a multi-stage cluster sampling approach was used to independently sample health facilities from within Ethiopia and Malawi. Sample size calculations were performed to estimate a precise point estimate of vaccine coverage. In Ethiopia, sampling occurred throughout five regions based on sample size calculations (estimated 32 000 records from 102 facilities) with a confidence level of 95%, precision of 5% and a design effect of three due to the multi-stage cluster sampling method. Within Malawi, sampling occurred within three regions based on sample size calculations (409 440 vaccination records from 42 facilities) with a confidence level of 95% and precision of 5%.Within Ethiopia, six data collectors extracted data from registration books into data extraction books that were prepared for each health facility. The registration books were scanned and the data collector submitted scanned images of the registration book and the data extraction book to the health centre at the end of the month. The data were double entered using Microsoft Excel and EPIinfo6-D. Within Malawi, six data collectors photographed vaccination records from paper-based child health registers. Data from photographs were double entered in Microsoft Excel or CSPro and exported to a customized Excel database.Within Ethiopia, data collection began in January 2012 and was completed in February 2013. Overall, a total of 102 health facilities were sampled to obtain the sample size of 1152 children. Within Malawi, data were collected at 41 facilities from January 2012 to April 2013 for PCV and April 2013 to January 2014 for Rota. During the data collection period, the catchment area of one of the 42 sampled health facilities in Malawi was consumed by another facility included in this study.Total administrations of the first dose of PCV (PCV1) and Rota (Rota1) were used as the main indicators of demand for the new vaccines. Children receiving vaccinations were categorized into the new birth cohort or backlog cohort based on their age at PCV1 and Rota1 launch. New birth cohort children were those who were eligible to start their routine immunization schedule as of the launch date (6 weeks old) or born any point after. Backlog cohort children receiving PCV1 were those who were older than 6 weeks but less than 1 year of age at the time of introduction. The small group of children who had no birthdate recorded were categorized into an ‘unknown’ group and children who were ineligible when they received the vaccination were categorized as ‘ineligible’; both of these categories were combined to create the cohort of ‘other’ children who received vaccinations. Due to more restrictive age eligibility requirements for Rota1 vaccination (up to 15 weeks instead of 1 year), in Malawi, the backlog cohort consisted of all children aged 6–15 weeks on the date of launch.To describe the pattern of demand, several key factors were characterized from the collected data. The months since launch were categorized based on the launch date for the facility in Ethiopia and on the national launch dates for PCV and Rota in Malawi. To characterize the number of people seen in any given day per facility regardless of vaccine type, the total number of children who received at least one vaccination per day per facility type was calculated. In Ethiopia, to characterize drop-out rates (proportion of children receiving PCV1 but not PCV3), children were categorized by their completion of the PCV series among all who obtained their first dose of PCV. The data collected from Malawi did not follow the same child for all three doses, and therefore, drop-out rates were not calculated.Both Ethiopia and Malawi vaccination records were weighted to account for unequal probability of selection and to obtain national-level estimates; for the few purposefully picked facilities, certainty units were created . The Cent-test comparison were calculated for the estimated population values. Penta was used as the benchmark in vaccine uptake as it was a well-established, routine immunization in both countries and its administration shares the same schedule as PCV for all three doses, and with Rota for the first two doses.To describe the relative demand for PCV1 and Rota1, the monthly doses of vaccines administered in the post-launch period were estimated and compared with the monthly doses of Penta1 (i.e. the first dose of Penta). Both the relative percent as well as the difference of means The relative composition of the demand of PCV1 and Rota1 was then examined by calculating the proportion and 95% confidence interval for the backlog demand over the total number of doses administered . To compare the differences in composition with respect to different age restrictions, the relative composition of demand was compared between PCV1 and Rota1 within Malawi for the same time periods by calculating the proportion of demand attributed to the backlog and the respective 95% confidence interval.t-test comparison were calculated for the estimated population values. Finally, to more thoroughly describe the age at which PCV1 and Rota1 were administered, age histograms were created for the backlog cohort and new birth cohort of children. The proportion of children receiving vaccines after 14 weeks was compared between the new birth and backlog groups using 95% confidence interval estimation.To inform EPI stock planning, we also calculated a planning ratio, which is the ratio of doses for PCV administered to the backlog cohort, ineligible children and children with unknown age vs the new birth cohort. Both the relative percent as well as the difference of means We then examined the possible implications of the magnitude of the total demand, by plotting the vaccinations administered per day per facility type over time, stratified by facility catchment size. We plotted the daily average facility size against the mean over month since launch to examine trends over time. Additionally, we illustrated the percent of children dropping out during the PCV series according to their age at vaccination within the backlog cohort.To account for the sampling design in variance estimations, a stratified multi-stage cluster method was declared using Stata 12.The estimates of the number of first doses of PCV and Rota administered post-launch as well as the composition of this demand are displayed in t-test P-value < 0.01] was 37% greater in Ethiopia. In Malawi, the 1-year relative demand for PCV1 [80.1 per 10 000 (SE 0.3)] compared with Penta1  was 59% greater; for the first 6 months, the relative demand of Rota1 [27.2 per 10 000 (SE 0.4)] was 5.9% greater than Penta1 demand .In the first year, the relative demand for PCV1 [290.4 per 10 000 (SE 9.4)] compared with Penta1  in Ethiopia; PCV1 [37.2 per 10 000 (SE 0.6)] was 188.8% higher than Penta1  in Malawi; and Rota1 [15.0 per 10 000 (SE 0.1)] was 15.4% higher than Penta1  in Malawi. In comparison, during Months 10–12 post-launch, demand for PCV1 [52.2 per 10 000 (SE 1.7)] vs Penta1  was 2.1% higher than Penta1 in Ethiopia and 1.1% higher in Malawi ; Rota1 [12.2 per 10 000 (SE 0.3)] demand was 4% lower than Penta1  demand in Malawi for Months 4–6 post-launch.During Months 1–3 post-launch, the demand for PCV1 [130.9 per 10 000 (SE 5.0)] was 112.8% higher than that of Penta1  in Ethiopia and 3.0 times higher in Malawi . This drops in later months similarly between the two countries with the final 3 month’s ‘non-new birth cohort’ demand [1.2 per 10 000 (SE 0.1)] 2.4% the size of the new birth cohort  in Ethiopia and 2.4% in Malawi . The ratio of backlog/ineligible/unknown administrations [7.0 per 10 000 (SE 0.01)] to new birth cohort administrations [20.2 per 10 000 (SE 0.1) t-test P < 0.01] was 35.0% for the Rota vaccine over the first 6 months after launch.The Ethiopia and Malawi experiences highlighted that demand for the newly introduced PCV and Rota was greater than for an established routine vaccine (Penta) in the year of introduction. Importantly, we found that this demand was particularly large in the first few months post-launch. A surge in uptake for NVI has been reported in the USA (This work found that the backlog cohort heavily shaped the pattern of demand observed in the first year. Although the WHO guidelines recognize the importance of two birth cohorts (i.e. the new birth and those still eligible to receive vaccinations based on age restrictions) when planning stock levels , the infConversely, we found evidence that uptake dynamics were affected by the age restrictions of the introduced vaccine. Specifically, the age restriction for the first dose of Rota1 was narrow (6–15 weeks) in Malawi when compared with that of PCV1 (any child 6 weeks–12 months old). This difference in age restrictions corresponded with unique patterns of demand. In the first 3 months post-Rota launch, the backlog demand for Rota was small—only 28.4% of the total demand, as compared with 71.5% for the PCV introduction. As a result, the excess demand for Rota as compared with the benchmark vaccine was only 15.4% higher.Although this smaller demand for Rota could be due to lesser social mobilization efforts rather than differing age restrictions, we observed that this was not the case. In fact, additional health care worker trainings and social mobilization activities were planned for the Rota vaccine launch compared with the PCV launch. Further, a study on caregiver knowledge, attitudes and practices conducted prior to the rotavirus vaccine launch found a high level of desirability, acceptability and interest in Rota, suggesting that caregivers did indeed wish to vaccinate their children . Thus, tWe found that the backlog cohorts for PCV1 in both Ethiopia and Malawi were comprised mainly of children over the age of 14 weeks. This finding highlights a potentially at-risk group of children who may not receive all of the three doses of PCV, as these older children do not have another scheduled vaccine dose to receive until 9 months of age (first dose of the measles vaccine). Because children must return to clinics ‘off schedule’ just to receive the full PCV dosing, they may be at greater risk of dropping out.These characteristics of demand composition should be taken into account when planning NVIs. Forecasting, stock planning, service delivery, reducing drop-out and monitoring can only be adequate by integrating strategies that rest on solid understanding of the backlog cohort’s demand.Countries with inclusive age eligibility criteria should request volumes of commodities  from suppliers based on forecast estimates that include the backlog cohort’s demand, if required. In addition, WHO should provide clearer recommendations to countries on the inclusion of the backlog cohort in their demand forecasts. This would allow donors such as Gavi, to revise their application materials to include more explicitly a forecast for the backlog cohort.Here we have seen that with PCV introduction in Ethiopia and Malawi, 37% and 59% more new vaccine doses were required compared with the benchmark routine vaccine (Penta) over the course of 1 year, primarily due to the backlog. For Rota in Malawi, only 5.9% more doses were required than for the routine vaccine over the first 6 months, likely because the age restrictions for the new vaccine were stricter.Thus, when setting age eligibility threshold for a new vaccine, countries should consider the impact of these guidelines on the size of the backlog cohort demand. Widening age eligibility on a new vaccine will lead to an increase in total demand for the vaccine (up to ∼60% more for PCV over the first year). Setting stricter age eligibility rules is an option to limit the financial and operational burden of NVI, but immunizing the backlog cohort is a significant opportunity to reach more children and accelerate herd immunity where epidemiologically relevant.If inclusive age eligibility restrictions are set, a large buffer stock for the new vaccine should be available beginning on the day of launch. Because demand from the backlog cohort is higher in the first months, failure to preposition additional stock would result in stock-outs.In Ethiopia and Malawi, excess demand outside the new birth cohort  for the new PCV was, respectively, 3.8 times and 3.0 times that of the new birth cohort in the first 3 months post-introduction. This ratio rapidly decreased, and excess demand was minimal by Month 7 in both Ethiopia and Malawi. The best practice is thus to preposition new vaccines at sub-national levels, e.g. district and facility levels, in the first months of introduction beyond the routine monthly allocation (up to four times more stock) ahead of launch. This may require putting in place temporary coping mechanisms if the cold chain capacity is restricted (e.g. a more frequent vaccine distribution schedule). Delivery schedules should align with the need to preposition stock, and contingency plans should also be put in place to allow for the allocation of transportation in the event of required emergency stock deliveries.Countries that intend to launch new vaccines with broader age eligibilty rules should prepare for increased service delivery needs at the time of introduction. Indeed, children who have already received routine vaccines come to the facilities at the same time as the new birth cohort. In Ethiopia and Malawi, immunization sessions were larger during the first month of PCV introduction: the average daily number of immunizations provided increased by 20% and 56% in Ethiopian health centres and Malawian hospitals, respectively. The average number of vaccinations provided remained higher than normal during the first 3 months of introduction, due to continued demand from the backlog cohort. Rota was introduced with narrower age restrictions, had a lower excess demand from the backlog cohort, and therefore, did not appear to have an impact on the average daily number of immunizations provided as observed at health facilities. Labour cost accounts for an important share of total costs for immunization. In their study on costing and financing of routine immunization and new vaccines (EPIC) in six countries, For future introductions with inclusive age restrictions, policy-makers should be mindful that excess demand from the backlog cohort may strain the health workforce, particularly in hospitals. Countries should thus carefully estimate whether the introduction of a new vaccine requires increasing human resource capacity at the health facility level during the first months following NVI, or whether the added workload can be absorbed by existing capacity. The need for increasing facility staff, which could also be a consequence of the widening of age eligibility criteria advocated in this manuscript, could negatively impact the cost-effectiveness of the new vaccines.Policy-makers planning future introductions must pay particular attention to the higher risk of drop-out of the backlog cohort. ‘Backlog’ children are out of sync with the official routine immunization schedule for the new vaccine. These children will require a greater number of visits to the health facilities to be fully immunized, as they typically do not benefit from the co-administration of the new vaccine for their second or third visit.We observed in Ethiopia that the drop-out rate for older children was higher than for younger children: over 35% of children aged 32 weeks or above did not complete the PCV schedule, as opposed to 20% of children aged 7–10 weeks. For future introductions, service delivery and social mobilization activities must mitigate this drop-out risk. Health care workers must be taught during pre-introduction training how to emphasize to caregivers of older children the need to come back to the health facility for additional visits. Social mobilization activities should also clearly underscore the number of doses required to be fully immunized, as well as clearly highlight age eligibility to reach children in the backlog cohort.Countries introducing new vaccines must update their data tools well ahead of the launch to ensure proper tracking of new vaccine delivery and consumption. After launch, countries should frequently monitor stock levels at the regional and district levels in the first 3–6 months. They should be ready to respond with emergency orders as needed.The Ethiopia and Malawi experiences highlight the need to closely monitor uptake dynamics to ensure against vaccine stock-outs, and to help the new vaccine quickly reach routine immunization coverage levels. In Malawi, this was done both via targeted phone calls to health facilities every 2 weeks and in-person supportive supervision visits. Due to the large backlog demand observed over the first months, this stock monitoring was essential to identify the need for additional stock in a timely fashion.More generally, replicating uptake studies such as the one we performed in Ethiopia and in Malawi offers an opportunity for policy-makers to better understand the profile of their countries’ demand for immunization, and thus better plan for the next introduction. Indeed, launching a vaccine should not be seen as an event but as a process, and the appropriateness of plans  can only be determined during the first year of introduction.Beyond vaccines, similar ‘backlog’ patterns are observed in other health areas that deal with age cohorts, such as guideline changes on CD4 count that impact viral load testing eligibility for HIV infection. For these areas, similar studies assessing uptake dynamics of new life-saving products would allow practitioners to better plan for their introductions, and thus maximize the opportunities they offer.The WHO’s Decade of Vaccines Collaboration Research and Development Working Group has outlined the need for targeted implementation research to improve the uptake of new vaccines . In partWhen planning an NVI, countries should consider the impact of the backlog cohort, especially in the first 6 months of introduction. Countries can maximize the life-saving potential of immunization through vaccinating a larger share of the backlog cohort, by setting looser age regulations and engaging in significant social mobilization. To cater to this backlog cohort, countries’ plans should request high stock volumes for the new vaccines, allocate adequate human resources and cold chain capacity to accommodate backlog cohort demand and include increased social mobilization activities to reach more children and reduce drop-out.


IgG4-related disease is rare, but a frequent differential diagnosis for malignant and for autoimmune diseases. Li and colleagues report the largest cohort of patients with IgG4-related sialadenitis. The observations reveal that the most important diagnostic step is obtaining biopsies. In addition, the IgG4 serum concentration may be a biomarker for the disease progression. Li and colleagues  a large hlebitis . Meanwhihlebitis . IgG4-RDWithin IgG4-RD, at least two major subgroups appear to be present: a subgroup with only head or neck involvement, which affects females and males equally; and a subgroup with either systemic involvement of many organs or of one organ outside the head and neck region, which affects mostly males (80 %) . Both foTo answer these questions on IgG4-RD, an exact diagnosis and therefore optimization of the diagnostic procedures is crucial. We thank Li et al.  for thein = 42) were carefully analyzed. Confirming earlier studies and different from patients with the systemic subgroup of IgG4-RD involving abdominal organs, females were more frequently involved than males. Concomitant autoimmune pancreatitis was observed in only 9 % of the patients, but the majority of the patients had a history of allergies. CT scans were more sensitive than the clinical examination in detection of enlarged salivary glands. In our opinion, ultrasound and magnetic resonance imaging (MRI) should be compared with CT scans in subsequent studies, since these techniques avoid radiation exposure, ultrasound would be easier to obtain, and MRI is currently the standard in assessing glandular structure.A large number of patients with IgG4-related sialadenitis  are helpful parameters in raising the suspicion of IgG4-RD, but similar to elevated IgG4 do not differentiate from malignancies. The most important diagnostic procedure is therefore a biopsy, which should be taken from a major salivary gland, preferably from the submandibular glands. In immunohistochemistry not only the previously defined features of IgG4-RD such as obliterative phlebitis or storiform fibrosis were identified, but also eosinophilia, present in 47 % of the patients, was helpful. Most important, biopsies can distinguish IgG4-related sialadenitis from malignancies of the glands, confirm the diagnosis, and justify the subsequent prolonged corticosteroid therapy.The findings of Li et al.’s study help to distinguish IgG4-related sialadenitis from Sjögren’s syndrome, which historically were considered to be related or even identical diseases. IgG4-related sialadenitis more often affects males and manifests as diffuse gland swelling rather than localized involvement of the parotid glands, the IgG4 is elevated, the presence of SS-A antibodies is rare, and histologically the plasma cells infiltrating the glands express IgG4 and the fibrosis is more pronounced as in Sjögren’s syndrome and storiform.This study by Li et al. is the largest so far with regard to histological analysis of the glands of patients with IgG4-related sialadenitis. Interestingly, the IgG4 serum concentration correlated with the glandular fibrosis and the reduction of saliva production and could therefore be a future biomarker for the disease progression. This is a new finding, which may be important in monitoring the efficacy of treatment of IgG4-related sialadenitis.Not all of the open questions on IgG4-RD can be answered by this study. The data give us information on diagnostic procedures and stress the importance of obtaining biopsies. The fact that most of the patients have an elevated IgE, a history of allergies, and eosinophilic infiltrates in the affected glands suggests an influence of interleukin-4 in the pathogenesis but does not yet prove an allergic rather than an autoimmune etiology of the disorder.