Early start of enteral feeding is an established treatment strategy in intubated patients in intensive care since it reduces invasive bacterial infections and length of hospital stay. There is equipoise whether early enteral feeding is also beneficial in non-intubated patients with cerebral malaria in resource poor settings. We hypothesized that the risk of aspiration pneumonia might outweigh the potential benefits of earlier recovery and prevention of hypoglycaemia.A randomized trial of early (day of admission) versus late (after 60 hours in adults or 36 hours in children) start of enteral feeding was undertaken in patients with cerebral malaria in Chittagong, Bangladesh from May 2008 to August 2009. The primary outcome measures were incidence of aspiration pneumonia, hypoglycaemia and coma recovery time. The trial was terminated after inclusion of 56 patients because of a high incidence of aspiration pneumonia in the early feeding group (9/27 (33%)), compared to the late feeding group (0/29 (0%)), p = 0.001). One patient in the late feeding group, and none in the early group, had hypoglycaemia during admission. There was no significant difference in overall mortality (9/27 (33%) vs 6/29 (21%), p = 0.370), but mortality was 5/9 (56%) in patients with aspiration pneumonia.In conclusion, early start of enteral feeding is detrimental in non-intubated patients with cerebral malaria in many resource-poor settings. Evidence gathered in resource rich settings is not necessarily transferable to resource-poor settings.ISRCTN57488577Controlled-Trials.com  Even with optimal antimalarial treatment with parenteral artesunate, mortality of severe malaria remains 15% in adults and 9% in children oIn the well equipped intensive care setting early start of enteral feeding is now common practice in a wide variety of patients, including those with sepsis We therefore performed an open randomized trial at a tertiary referral hospital in Chittagong, Bangladesh to compare early versus late start of nasogastric tube feeding in patients with cerebral malaria. Incidences of aspiration pneumonia, hypoglycaemia and coma recovery time were the primary outcome measures.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see This study was conducted according to the principles expressed in the Declaration of Helsinki. Ethical clearance for the study was obtained from the Ethical Review Committee of Chittagong Medical College, and from the Oxford Tropical Research Ethics Committee (OXTREC). ISRCTN registration number ISRCTN57488577. For all participants, prior written informed consent was obtained from an attending relative.The study was conducted at Chittagong Medical College Hospital (CMCH), Chittagong, Bangladesh from June 2008 to August 2009. CMCH is a 1000-bed teaching hospital with limited facilities for intensive care, oxygen therapy, blood transfusion, and renal dialysis. Provision of optimal nursing care is sometimes problematic because of the large patient load. Malaria transmission around Chittagong is seasonal and of low intensity.Adults and children ≥2 years with slide proven cerebral Plasmodium falciparum malaria  were included in the study, provided that written informed consent was obtained. Patients with features of aspiration pneumonia on admission, based on an abnormal chest examination and pulse oximetry and/or chest x-ray findings were excluded. Other exclusion criteria were: pregnancy, diabetes mellitus requiring insulin or contraindications to enteral feeding , severe vomiting (>6 times per 24 hours) or severe dehydration, pancreatitis (laboratory confirmed: serum amylase >500 U/L), or children with severe malnutrition (according to WHO criteria)) On enrolment, a full history and physical examination were performed. Nutritional status of the patient was assessed by skin-fold thickness in the triceps region of the left arm, mid upper arm circumference, weight, height and presence of bilateral pedal oedema.R) when the patient had recovered sufficiently to eat and take tablets.Patients were managed in accordance with the World Health Organization guidelines for malaria 2006 ), or no enteral feeding until able to take oral food or up to a maximum starvation period of 60 hours in adults and children >12 years old and 36 hours in children ≤12 years old (‘late feeding’). The randomization code was generated by a statistician at the Mahidol-Oxford Research Unit in Bangkok. Treatment allocations were kept in consecutively numbered sealed envelopes and opened by the research physician upon enrollment of the patient into the study.All patients received a nasogastric (NG) tube on admission. Patients were randomized in advance in blocks of 20 by an independent investigator to either receive enteral feeding upon admission through the NG tube  . This was dissolved in bottled water according to the instructions of the manufacturer providing 1 kcal per ml feed.The position of the NG tube was checked by a study doctor before starting the first feed by injecting air with a large syringe and listening over the abdomen, and before subsequent feeds by patient attendants who had been specifically trained by the study doctor by checking the position of a mark drawn on the tube at the time of insertion and by aspiration of gastric contents. The head of the bed was elevated to 30°–45°, where possible throughout the admission, otherwise for a minimum of 30 minutes after feeding followed by placing the patient in the recovery position. NG feeding for adults was started at a rate of 2–4 ml/kg feed given as a slow bolus every 2 hours with 10 feeds per day, omitting 2 late night feeds corresponding to delivery of 1000–2000 kCal per 24 hours to an adult weighing 50 kg. For children, the volume of enteral feeding was calculated by subtracting the amount given intravenously from the total amount of fluids required per 24 hours, based on age and body weight: . The total amount of enteral feed in children was divided into 12 feeds, given every 2 hours round-the-clock.rd of the volume of the previous feed in children. If gastric retention was present, feeding was withheld and a prokinetic drug was administered (domperidone suppositories 20mg every 8 hours for adults and 15mg (1/2 suppository up to 5 years & 1 suppository over 5 years of age) for children). If gastric retention was not present, any aspirated feed in the syringe was discarded and feeds were continued every 2 hours according to schedule.The volume of aspirated gastric content was measured 2-hourly just before every feed. Gastric retention was defined as >4 ml/kg in adults and >2/3Adult patients (>12 years) in the late feeding group received 50–60 ml 5% dextrose-saline/kg/24 hours intravenously with an energy content of around 200 kCal/L, corresponding to 10–12 kCal/kg/24 hours. Paediatric patients (≤12 years) in the late feeding group received intravenous 10% dextrose in 0.45% saline in the volumes stated above until feeding was started. This contained 400 kCal/L corresponding to: 40–48 kCal/kg/24 hours for those aged 2–4 years, 36–40 kCal/kg/24 hours for 4–8 year olds and 24–28 kCal/kg/24 hours for 8–12 year olds.As in many hospitals in the tropics, very few nursing staff were available (around 5 nurses for up to 100 patients) and much of the basic nursing care was provided by family members. The administration of enteral feed was performed largely by family members, with the support of the nursing staff and study doctors. Family members were instructed in the techniques for checking NG tube position using a pre-drawn mark, checking for gastric retention and giving NG feeds.o–45o head tilt position whenever possible and turned on their other side every 2 hours. Fluid input and output was closely monitored. If needed in the comatose patients, maintenance of an open airway was secured with a Guedel.Patients were monitored 4-hourly by a study doctor for GCS or BCS, blood pressure, respiratory rate, temperature, oxygen saturation by pulse-oximetry, evidence of aspiration and signs of aspiration pneumonia. Patients were nursed in a 30On admission a venous blood sample was taken for parasitaemia, complete blood count, pH, bicarbonate, blood standardized base excess, glucose, electrolytes and blood urea nitrogen. Peripheral blood parasitaemia in thin or thick film, as appropriate, was assessed on admission, followed by 12 hourly sampling until complete parasite clearance, defined by 2 consecutive negative peripheral blood slides. Capillary blood glucose was checked 4 hourly until the patient regained consciousness. A blood glucose level ≤2.8 mmol/l was treated with 10% intravenous glucose (4 ml/kg body weight). Repeat blood count and biochemistry and blood cultures were done when indicated.Aspiration pneumonia was diagnosed clinically from history and chest examination , and confirmed by chest X-ray (CXR). All patients had a CXR upon admission. The CXR was repeated upon indication and 3 days after the first one for follow-up. Treatment of aspiration pneumonia was with antibiotics (intravenous ceftriaxone plus metronidazole). Oxygen was given by nasal cannula or face mask where indicated. In the case of severe respiratory insufficiency, the patient was referred to the intensive care unit of CMCH for mechanical ventilation if a bed was available.9/l or ≤4×109/l, or a differential count showing >10% immature neutrophils. Other secondary outcome measures were the time to sit independently, time to speak, time to eat independently, total duration (days) of admission in the hospital and in-hospital mortality.The primary outcome measures were the incidence of aspiration pneumonia, hypoglycaemia (<2.8 mmol/L) and coma recovery time . Incidence of sepsis was a secondary outcome measure. Sepsis was defined as the presence or suspicion of infection  plus systemic inflammatory response syndrome (SIRS) as indicated by ≥3 of the following criteria: prolonged fever i.e. axillary temperature ≥38°C or core temperature of ≤36°C; heart rate of ≥90 beats/min; a respiratory rate of ≥20 breaths/min (up to 5yrs ≥40) or the use of mechanical ventilation for an acute respiratory process; a white-cell count of ≥12×10A sample size of 62 in each treatment group was calculated in order to detect an increase in incidence in aspiration pneumonia from 10% to 30% with alpha of 0.05 and power of 0.8.Statistical analysis was performed by SPSS software (version 15.0) and STATA (version 10). When appropriate, data were log transformed to obtain a normal distribution. Normally distributed data were compared using Student’s t test. The Mann-Whitney U test was used for nonpaired nonparametric data. Categorical data were compared by Pearson’s chi-squared test or Fisher’s exact test, as appropriate. The level of significance was p<0.05.Adverse events were reported to an independent data safety monitoring committee . An interim analysis was planned and done after enrollment of 50 patients. Prespecified stopping rules for this interim analysis were a statistically significant difference between the groups in aspiration pneumonia, hypoglycaemia or mortality. On advice of the committee the trial was stopped on 2nd September 2009, because of the high incidence of aspiration pneumonia in the study group receiving early enteral feeding.The study was externally monitored to assure compliance with the International Conference on Harmonisation of Good Clinical Practice guidelines.A total of fifty six patients with cerebral malaria were enrolled and randomized to receive early (n = 27) or late (n = 29) start of enteral feeding . Of thesAspiration pneumonia occurred in 9/27 (33% (95% CI 18–52%)) patients who started early nasogastric tube feeding, compared to none (0% (95% CI 0–14%)) in those in the late feeding group ) in the late feeding group feeding, compared to none (0% (95% CI 0–15%)) in the early group . The geometric mean (95% CI) volume of feed given per patient was 2.7 (1.6–4.7) L over a median (quartiles) of 2 (1–3) days.There was no difference in coma recovery time between the two groups .There were no differences in incidence of sepsis, times to sit, speak or eat independently, duration of admission or overall mortality between the two groups . Median Start of early enteral feeding is an established and evidence based strategy in critically ill patients in the intensive care unit. However, the evidence for this practice is obtained in well-resourced Western ICU settings, whereas the strategy is also followed in many hospitals in resource-poor settings where endotracheal intubation is not generally available. Early start of nasogastric tube feeding was routine practice in CMCH at the moment the current study was instigated. We showed that almost a third of cerebral malaria patients receiving early enteral feeding on the first day of admission developed aspiration pneumonia, whereas no patient in the control group did. Although there was no difference in overall mortality between those who received early and late enteral feeding, there was an excess mortality in those with aspiration pneumonia. Previous studies have shown that early enteral feeding can reduce mortality Vomiting and gastric retention were not identified as risk factors for aspiration pneumonia in this study, which is in contrast with previous studies In accordance with normal practice in many hospitals in the region, much of the nursing care, including administration of enteral feeding, was provided by patients’ relatives rather than trained staff. Although they were carefully instructed, and volumes of feeds were carefully monitored, adherence to the study procedures by these family members could not routinely be verified. It is possible that the incidence of aspiration may have been lower if more staff had been available to assist with care. Another shortcoming was that in this unblinded randomized study, the assessment of aspiration pneumonia was supported by positive findings on the chest X-ray in only 3/9 patients, whereas in 6/9 patients an X-ray could not be obtained. However, findings on physical examination were clearly compatible with pneumonia in all these cases and pneumonia only occurred in the early feeding group following witnessed aspiration after commencing feed thus leaving little room for alternative diagnoses.Most patients developing aspiration pneumonia aspirated during the first 24 hours of admission, while still being unconscious, and they had a lower median GCS than those who did not aspirate. This is in agreement with other studies describing increased risk of aspiration pneumonia in patients with a GCS<9 In conclusion, this study provides evidence that early enteral feeding is detrimental in patients with cerebral malaria treated in resource poor settings where endotracheal intubation is not generally available. Early enteral feeding increases the risk of aspiration pneumonia and conveys no clear benefits. Evidence gathered in resource rich settings is not necessarily transferable to resource-poor settings.Protocol S1Study protocol.(DOC)Click here for additional data file.Checklist S1CONSORT 2010 checklist.(DOC)Click here for additional data file.


The purpose of this study was to compare the mechanical properties, host responses and incorporation of porcine small intestine submucosa (PSIS) and porcine acellular dermal matrix (PADM) in a rat model of abdominal wall defect repair.Prior to implantation, PSIS and PADM were prepared and evaluated in terms of structure and mechanical properties. Full-thickness abdominal wall defects were created in 50 Sprague-Dawley rats, and were repaired using either PSIS or PADM. Rats were sacrificed 1, 2, 4, 8 and 12 weeks post-repair and examined for herniation, infection, adhesions, contraction, and changes in the thickness and strength of the tissues incorporated at the defect sites. Histopathology and immunohistochemistry were performed to analyze inflammatory responses, collagen deposition and vascularization.P<0.01). However, the mechanical properties observed after integration with the surrounding native tissues was similar for PADM and PSIS. Both PADM and PSIS showed significant contraction by week 12. However, PADM tissue induced less adhesion and increased in thickness more slowly, and showed less infiltration by foreign giant cells, polymorphonuclear cells, and mononuclear cells. Improved remodeling of host tissue was observed after PSIS implantation, which was apparent from the orientation of bands of fibrous connective tissue, intermixed with newly formed blood vessels by Week 12.PADM showed more dense collagen deposition and stronger mechanical properties than PSIS prior to implantation (PSIS showed weaker mechanical properties prior to implantation. However, after implantation PSIS induced more pronounced host responses and showed better incorporation into host tissues than PADM. Currently, more than 70 types of mesh are available, and are classified as synthetic material or biological material according their particular composition in vitro and to compare their mechanical properties, host responses and tissue incorporation after implantation in vivo.Porcine small intestine submucosa (PSIS) and porcine acellular dermal matrix (PADM) are two naturally-derived materials used for biological meshes that have been used clinically. PSIS is prepared from the submucosa of the small intestine of pigs in a manner that removes all cells, but retains the natural 3-dimensional (3D) composition of the extracellular matrix (ECM). The ECM acts as a scaffold into which cells can migrate and proliferate. PSIS has a high collagen  content that forms the scaffolding for the ECM. The non-collagenous portion within PSIS contains numerous growth factors such as FGF-2, TGF-β and VEGF Fifty male Sprague-Dawley rats, each weighing 200–250 g, were obtained from SLAC National Rodent Laboratory Animal Resources . All animal study protocols were approved by the Institutional review committee of Shanghai Jiao Tong University School of Medicine (ID: SYXK 2008-0050). Rats were housed in accordance with current national guidelines regarding animal welfare. The environment was maintained at 18–26°C with a relative humidity of 30–70%.60Co, Shanghai Institute of Applied Physics, Chinese Academy of Sciences). The two-layer PSIS scaffolds were trimmed to a size of 50×40×0.2 mm3 prior to implantation. Samples were stored at −80°C until required.Fresh porcine small intestine was obtained from Fuxin abattoir . Segments of fresh porcine small intestine were cut into lengths of approximately 5 cm and flushed through with tap water to remove the intestinal contents. After longitudinal splitting of the intestinal segments, the serosal and muscular layers were removed mechanically and de-cellularized. This was done by shaking the small intestinal segments at 4°C in 1 L of 0.2% TritonX-100  containing 26.5 mmol/L ammonium hydroxide for 7 days. After de-cellularization, the small intestinal segments were washed in deionized water for 72 h. The resulting PSIS was freeze-dried at −55°C for 48 h and sterilized using γ-rays .Fresh porcine skin was obtained from Fuxin abattoir. After a thorough cleaning, excision of the sub-dermal fatty tissue and hair removal, the resulting derma was cut into pieces. The pieces were soaked in a solution containing 0.25% trypsin solution at 4°C for 24 h, followed by treatment with a 0.1% SDS solution at room temperature for 6 h, and further treatment with 560 units/L of Dispase solution at 4°C for 24 h. Finally, the tissue was treated with 0.1% SDS solution at room temperature for 6 h before being washed twice in PBS buffer for 30 min. The porcine derma was then cross-linked using glutaraldehyde to prevent rapid degradation and 50×40×0.5 mmSamples were freeze-dried, loaded onto aluminum studs and coated with a thin layer of gold for 3 min at 8 mA at 0.1 Torr. The collagen morphology was then examined under a scanning electron microscope . Samples were scanned and the micrographs recorded. The morphological arrangement of the collagen fibers in the two materials after acellular treatment was compared.2 full-thickness defect, including the fascia, muscles and peritoneum, was created centered on the midline. The rats were then randomly assigned into PSIS and PADM groups (n = 25) and a 50×40 mm2 section of sterile PSIS or PADM was implanted into each rat to repair the abdominal wall defect. Each side of the scaffold overlapped the edge of the defect by 10 mm and was fixed using 3-0 polypropylene interrupted sutures. The skin was then closed using 2-0 Vicryl interrupted sutures. No antibiotic treatment was given during the experiments.Each Sprague-Dawley rat was anesthetized using an intramuscular injection of ketamine (60 mg/kg). The abdominal skin was shaved and disinfected with a povidone iodine solution. A midline incision, 50 mm in length, was performed along the linea alba and a 30×20 mmFive rats from each group were randomly sacrificed 1, 2, 4, 8 and 12 weeks after implantation. Any evidence of seroma, hematoma, or infection of the implants on the subcutaneous and visceral sides was noted upon gross examination before sampling. The implant dimensions were measured using a centimeter scale and recorded to assess any subsequent intra-corporal contraction. The abdominal wall was distended with 200 ml saline and any abnormal abdominal protrusion, before or after mechanical examination, was considered to be a fascial weakness or a hernia Serial sections of the explants (the implant plus 1 cm of the surrounding tissues and the interface) were embedded in paraffin, cut into 3 µm sections and stained with hematoxylin and eosin. Microscopic evaluation was performed to quantify the number of foreign body giant cells, polymorphonuclear cells (PMNs) and mononuclear cells (MNs) invading the implanted scaffolds. Qualitative assessment of collagen deposition was performed using the Masson trichrome stain. The amount of inflammatory cell infiltration and the amount and organization of any collagen deposits were scored using a method analogous to that described by Badylak bv) was calculated for 10 randomly selected high-contrast fields at ×200 magnification using Image-Pro Plus (v. 6.0) as follows: percentage area of blood vessels = area of capillary vessels/total tissue area. All evaluations were performed by one pathologist who was blinded to the materials being tested.Immunostaining was performed to assess any neovascularization of the implants. Samples were incubated with an anti-CD31 antibody  for 60 min. Sections incubated without the primary antibody served as negative controls. The mean percentage area of blood vessels . The length of the tested sample, held between two grippers, was set at 25 mm. Samples were stretched along their longitudinal axes at a cross-head speed of 10 mm/min until failure. The load and displacement were recorded throughout the elongation and converted to a stress-strain curve based on the initial specimen dimensions. The stiffness (N/mm) was determined by calculating the slope of the load (linear portion) vs. displacement. Five samples of each material were tested.Mechanical examination of the 50×10 mmThe bursting strength of the materials was examined using a bursting strength tester  according to ASTM Standard The load on the explants was measured as describe above. All of the explants tore at the interface during tensiometry testing. The reported values do not actually represent the strength of the material, but rather represent the degree of integration at the interface with the surrounding tissues. This is known as the “strength of incorporation” P<0.05 was determined to be statistically significant.All data were expressed as the mean ± SD. The difference between PSIS and PADM in terms of mechanical properties, gross evaluation and host response were analyzed using the Student's t-test. The statistics package for social science  software was used for statistical analysis. PSIS and PADM were successfully prepared as shown in P<0.01). However, PSIS had better extension properties than PADM (P<0.01).PSIS and PADM were tested for maximal loading, maximal displacement, stiffness and bursting strength . The meaAll rats recovered normally from surgery and survived to their predetermined sacrifice date. None of the rats showed any evidence of bulging or herniation at the implantation site either before or after instillation of 200 ml saline. Seroma were noted in two cases in the PSIS group and four cases in the PADM group 2 weeks after implantation. Intestinal obstruction was noted in one case in the PADM group 2 weeks after implantation. Further gross examination showed evidence of a small intestinal obstruction secondary to an adhesion, resulting in a kink in the intestine at the site of the central portion of the implant. There were no clinical signs of hematoma or infection in either group.2) and PADM (12.6±3.1 cm2) groups 12 weeks after implantation than it was prior to implantation (20 cm2) (P<0.01). The contraction rate was 29% for PSIS and 37% for PADM. There was no significant difference in surface area contraction between the PSIS and PADM groups after implantation , and the tenacity of the adhesions and surface area scores in the PSIS group were almost 4-fold those in the PADM group. Two weeks after implantation, the level of adhesions in the PSIS group decreased dramatically, and was not significantly different from that in the PADM group. Meanwhile, the adhesions in the PADM group were mild, and decreased slightly between 2 to 12 weeks after implantation . The increase was significantly higher in the PSIS group than that in the PADM group between 2 and 12 weeks after implantation  and PADM (0.8±0.05 mm) groups than it was prior to implantation . These inflammatory reactions gradually became weaker and fell to negligible levels (similar to those in PADM rats) by 12 weeks after implantation (P>0.05). Overall, the PADM rats showed substantially lower levels of inflammatory cell infiltration over the 12 weeks  appeared 1 week after implantation. Rats in the PSIS group showed a pronounced inflammatory response 1 to 4 weeks after implantation, which was significantly greater than that seen in the PADM rats (12 weeks .P<0.05). Lower levels of blood vessel ingrowth were observed in PADM rats up until Week 12. The density of capillary ingrowth, as indicated by %Abv, was significantly higher in the PSIS group (5.2±1.3%) than that in the PADM (2.6±0.7%) group during the entire experimental period . There was no significant difference in collagen organization between the PSIS and PADM groups after implantation (P>0.05; Masson trichrome staining showed that PSIS induced more intense collagen deposition from 2 to 12 weeks than PADM ( P>0.05; .P>0.05; The strength of incorporation of the PSIS and PADM explants decreased in the first 2 weeks, and then increased gradually over the following 10 weeks. There was no significant difference between the maximal loads borne by the PSIS and PADM explants P>0.05; .Several xenogeneic biological materials, including bovine dermis, bovine pericardium, ovine dermis, PSIS and PADM, are available for the tension-free closure of abdominal wall defects in cases where a synthetic mesh is not indicated or where inadequate amounts of autogenous musculofascial tissue are present in vivo was the optimal method to evaluate the mechanical properties for two groups which was limited by operation in this study. An increase in the thickness of the implants appears to be necessary to maintain body abdominal wall integrity during remodeling and biological degradation of the scaffold. Our results showed that both PSIS and PADM were gradually replaced by connective tissue; developing a thicker abdominal wall layer by the end of the observation period. This increase in implant thickness was particularly evident for PSIS, which is consistent with the greater inflammatory responses observed with this implant. Contraction of the implants may reflect an inadequate rate of vascularization, leading to inadequate nutrition and subsequent necrosis and fibrosis The implantation of PSIS, an acelluar collagen-based biodegradable matrix derived from the submucosal layer of the porcine intestine, resulted in minimal immune responses in rats. PADM, isolated living cells and non-collagenous material, represents intact porcine dermal collagen and retains the original 3D structure of the dermis. Each of these materials is characterized by its own distinctive physical, mechanical, and biological properties. Our results demonstrated that both PSIS and PADM maintained sufficient strength and incorporate host tissues to repair abdominal wall defects efficiently. There was no evidence of bulging or herniation at the defect sites in either group during the post-operative observation period, even after instillation of 200 ml saline into the peritoneal cavity. The test of infiltrating saline was carried out to evaluate the properties of implants that response to the burst increasing of intra-abdominal wall pressure. The results revealed that the increase in intra-abdominal pressure still did not exceed abdominal wall counterpressure after implantation, though testing the entire implants using bursting strength tester Further histological evaluation showed that the host responses and incorporation of PSIS were different from those observed from PADM. PSIS implants caused a more pronounced inflammatory response, as evidenced by infiltration by both PMNs and MNs during the initial stages; however, this inflammatory response rapidly diminished to a level similar to that seen in PADM implants, which induced a less severe inflammatory response throughout of study period. Compared with the PADM implants, the PSIS implants degraded more quickly and were almost totally replaced by organized collagenous tissues. Little PSIS material remained after 12 weeks, which suggests a better remodeling process. This was supported by the finding of prominent, well-vascularized and organized fibrous connective tissues with PSIS, in contrast to the lack of well-vascularized fibrous connective tissue orientation seen with PADM. The original PSIS scaffold material was no longer evident. Instead, well-organized, oriented bands of fibrous connective tissue were present.These results demonstrate that the use of PSIS results in better host incorporation and remodeling in terms of the amount and organization of the deposited collagen, improved neovascularization, a looser 3D meshwork and growth factors contained, all of which may promote host tissue repairing and remodeling compared with PADM. These results are consistent with those of Mattia et al, which focused on commercial PSIS and PADM in vivo studies showed that the strength of PSIS was similar to that of PADM, and there was no significant difference in the maximal loads borne by PSIS and PADM after implantation. PSIS possesses a “looser” structure than PADM which has a thicker and denser collagen arrangement. However, although the cross-linking is believed to stabilize the implant by preventing degradation by collagenases, they may also change a potentially biologically interactive material into a relatively inert material, preventing repopulation and remodeling by host cells The strength of incorporation is a well-established measurement of the incorporation of host tissues into an implant in vivo. Our study shows that PSIS is a viable alternative to existing scaffold materials for the repair of abdominal wall defects, and elicits a distinctive host tissue response. Apparently, the 2-layer PSIS we prepared may possess insufficient mechanical strength needed for the clinical study. The commercial biomaterial of PSIS are much thicker to provide adequate strength for the repair of abdominal wall defects owing to its multi-layered and cross-linked structure at the costs of moderate host incorporation in vivo. Further long-term studies are needed to determine the mechanical changes, incorporation into adjacent soft tissues and techniques to reduce the development of adhesions. The balance will be maintained by using various multi-layered and non-cross-linked PSIS. Furthermore, the application of tissue engineering technology to PSIS may be helpful in the development an ideal abdominal wall defects repair material.In summary, our data suggest that both PSIS and PADM were well tolerated for a period of 12 weeks following implantation into Sprague-Dawley rats. PSIS was superior to PADM with respect to implant thickness, host incorporation including vascularization and collagen deposition, which all correlated well with the materials physical properties. Although the mechanical properties of the PADM scaffold were superior to those of PSIS, there was no significance difference in the strength of incorporation between these two materials


Incident irradiance exponentially attenuated in the same zone reaching 1% at 1.7-mm depth. Proteobacteria dominated all layers of the mat (24%–42% of the identified bacteria). Anoxygenic photosynthetic bacteria (dominated by Chloroflexus) were most abundant in the third red layer of the mat (L3), evidenced by the spectral signature of Bacteriochlorophyll as well as by sequencing. The deep, black layer (L4) was dominated by sulfate reducing bacteria belonging to the Deltaproteobacteria, which were responsible for high sulfate reduction rates (measured using 35S tracer). Members of Halobacteria were the dominant Archaea in all layers of the mat (92%–97%), whereas Nematodes were the main Eukaryotes (up to 87%). Primary productivity rates of Um Alhool mat were similar to those of other hypersaline microbial mats. However, sulfate reduction rates were relatively low, indicating that oxygenic respiration contributes more to organic material degradation than sulfate reduction, because of bioturbation. Although Um Alhool hypersaline mat is a nutrient-limited ecosystem, it is interestingly dynamic and phylogenetically highly diverse. All its components work in a highly efficient and synchronized way to compensate for the lack of nutrient supply provided during regular inundation periods.The Um Alhool area in Qatar is a dynamic evaporative ecosystem that receives seawater from below as it is surrounded by sand dunes. We investigated the chemical composition, the microbial activity and biodiversity of the four main layers (L1–L4) in the photosynthetic mats. Chlorophyll  The activities of photosynthetic microorganisms (oxygenic and anoxygenic), aerobic heterotrophs, and sulfate reducers in addition to mass transfer limitation lead to the formation of distinct microenvironments. These dynamic microenvironments vary on both spatial and temporal scales providing heterogeneous environments leading to diverse community structures.Because of limited ability to isolate the majority of microorganisms, the application of culture-independent approaches to the study of microbial ecology, in general, and in microbial mats in particular has expanded our understanding to this field The coastal ecosystems of Qatar and those of the neighboring Gulf countries are found in one of the most arid regions of the world. These coastal ecosystems are fragile and threatened by human activities such as urban development, pollution, oil and gas mining and uncontrolled recreation. The coastline in Qatar is generally flat, and mangroves are locally a prominent feature. Coastal Sabkha are predominantly found on the eastern coast of Qatar, with the largest site located in the southeast, immediately south of Mesaieed Sabkha. Mesaieed is the largest coastal Sabkha in the country, extending over 55 km in length and between 4 and 14 km in width. Apart from saline crust, Mesaieed Sabkha is characterized by high gypsum content in the subsurface layers. The texture of these layers can be characterized as sandy clay-loam. Um Alhool is part of Mesaieed Sabkha and is located between Al Wakra and Mesaieed at south east of Qatar  that seeps from the adjacent Arabian Gulf water from below in addition to limited input from rain (never exceeds 100 mm y−1). The mat was covered by approximately 30 cm of sea water with water temperatures ranging between 15–26°C in December; however, water temperature can reach more than 33°C (air temperature > 47°C) in summer. The mat is also inundated with seawater in summer, but it is shallower and has higher salinity (>13%) than in winter because of high evaporation rates.Um Alhool is part of Mesaieed Sabkha, which is the largest Sabkha in Qatar. It is localized at south east of Qatar . The Um in situ conditions. All the required permissions for visiting the site and taking samples were issued from ESC, who is partner in this work. No sampling for vertebrates was done in this study. Microsensor measurements and DNA extraction were performed on those samples one day after collecting them. Additional mat samples were sent to the Max-Planck Institute for Marine Microbiology, Germany, where they were incubated under similar conditions . The mat pieces were kept under a 10-h light–14-h dark illumination regime at incident irradiance of approximately 480 μmol photon m−2 s−1 with a spectral composition similar to sunlight. Pigment analysis and sulfate reduction rates were conducted in the Max-Planck Institute for Marine Microbiology.Microbial mat samples (10×20 cm) were collected in December 2011. Several mat pieces were taken from the field, transported in the ambient sea water to the Environmental Study Center (ESC), Qatar University, and kept under simulated Surface and pore waters were collected from the study sites in December 2011. Pore waters were taken at depths of 1, 3, 5, 8, 10, 13, and 15 cm below surface with about 20-cm-long pore water zippers made of stainless steel lances connected to plastic syringes via 3-way valves and immediately filtered through 0.45-μm disposable membrane filters into pre-conditioned different sampling vials: (i) samples were collected in plastic centrifuge tubes containing 5% zinc acetate solution for sulfate and sulfide, (ii) acid pre-cleaned glass exetainers for dissolved inorganic carbon, and (iii) tubes without additions were used for nutrient samples see . AliquotSilicate, phosphate, ammonium, nitrate, and nitrite were spectrophotometrically quantified with a Skalar continuous-flow-analyzer according to methods previously described a in the mat was approximated from the hyperspectral image by calculating the hyperspectral microphytobenthos index (MPBI) Pigments concentrations were semi-quantitatively measured using hyperspectral reflectance imaging of the intact mat piece and quantitatively using High Pressure Liquid Chromatography (HPLC) on pigment extracts. Hyperspectral imaging and photopigment identification from the hyperspectral images were performed as previously described λmaxR is the measured reflectance at maxλ and pR is the value of the fitted linear trend in the NIR range (720–800 nm) extrapolated to λmax.where Rhodopseudomonas sphaeroides  was used, this pigment was quantified only in arbitrary units. The scytonemin standard was from Sigma-Aldrich, Germany and the other standards are from DHI Water and Environment, Denmark.The uppermost 6 mm part of the mat were sliced in 0.5-mm thick layer for the first 3 mm and 1 mm for the rest using cryomicrotome  at −36 °C. Pigments were extracted by adding 1 ml cold acetone (100%) on top of the freeze-dried slices, which were then incubated in sonication water bath for 3 min, and then they were kept in dark at −20 °C overnight. Subsequently, the supernatant was filtrated through a 0.45-μm Acrodiscs CR 4-mm syringe filter  and the filtrates were injected into a reverse-phase HPLC consisting of a Waters 996 photo diode array detector (PDA) and a Waters 2695 separation module . Pigments were separated on the basis of the method described by Wright et al. A microbial mat sample was placed in a small flow-chamber (11×4.5×5 cm) connected with plastic tubing fixed to a submersed water pump to maintain steady flow of seawater  above the mat surface. The flow cell was fixed on a holder and placed under a vertically incident collimated light beam from a tungsten-halogen lamp .2 concentration were performed with a fast response Clark-type microelectrode , as well as volumetric rates of gross photosynthesis  was calculated as Rλ  =  Iλ,mat/Iλ,ref, where Iλ,mat and Iλ,ref are the back-scattered spectral radiances measured above the mat and a white reflectance standard , respectively, with a fiberoptic field radiance microprobe −2 s−1) was measured by a PAR quantum irradiance sensor (LI-190 Quantum) connected to a light meter .Light attenuation in the mat was measured using a fiber-optic scalar irradiance microprobe (80 μm) 2 and scalar irradiance microsensors were positioned at zenith angles of 135° and −135°, respectively, relative to the vertically incident light. The measuring tips of both microsensors were positioned at the mat surface and in close proximity to each other using a 3-axis manual micromanipulator aided by observation under a dissection microscope .To prevent self-shading and allow simultaneous measurements, the O35S-labeled sulfate (≈50 kBq) in each layer (1 cm thick) as previously described 35S-labeled-reduced inorganic sulfur was determined using the single-step cold distillation method of Kallmeyer et al. −3 d−1) were calculated using the equation described in Microbial sulfate reduction rates were measured using the whole-core incubation technique by the incubation of short sediment cores (2.6 cm diameter) after the injection of 20 μl of carrier-free Distinct layer of the microbial mat, based on color (2–3 mm), were sliced using sterile scalpels after bringing the samples from the field to the lab. DNA was extracted and purified from each layer using the UltraClean soil DNA isolation kit  according to the manufacturer’s instructions.AACCTGGTTGATCCTGCCAGT and Euk570R GCTATTGGAGCTGGAATTAC) for eukaryotic sequences. Pyrosequencing was performed by Research and Testing Laboratories, Lubbock, Texas, using a Roche 454 FLX Genome Sequencer system as previously described by Dowd et al. The extracted DNA from the layers was then analyzed by 454 pyrosequencing for bacterial, archaeal and eukaryotic diversity. Primer sets (27Fmod AGRGTTTGATCMTGGCTCAG and 519Rmodbio GTNTTACNGCGGCKGCTG) were used for bacterial sequences, (archea349F GYGCASCAGKCGMGAAW and archaea806R GGACTACVSGGGTATCTAAT) for archaeal sequences, and  and phycocyanin (625 nm) detected by hyperspectral imaging  of variable thickness (1–2 mm) at the mat surface see . This di imaging . However imaging , which i imaging  were scaDirectly underneath the green layer, a brown layer (L2) of ∼1.5 mm followed by a very thin (∼1 mm) red layer (L3). The latter layer was permanently present in the whole mat ecosystem and not just in this specific piece. The red layer is characterized by the presence of anoxygenic phototrophic microorganisms evidenced by pronounced absorption at the wavelengths of 778 and 830 nm .In addition, the mat ecosystem has ample eukaryotic microorganisms and mesofauna that can be seen by microscope and by eyes, respectively. For example, several worms and small marine arthropods were seen at the surface of the microbial mat in the morning, because of OPotassium and phosphate concentrations in Um Alhool mat were higher than those characterizing marine water. Potassium and phosphate concentrations in Um Alhool mat were 20 and 0.8 μM, respectively, compared with those of 10 and 0.53 μM in seawater . Howevera and bacteriochlorophylls. Chl a was detected in all mat layers . Bacteriochlorophyll concentration  and gradually decreased with the depth, until it was below the detectable limit of <4 mm. The highest concentration of fucoxanthin , was found 1 mm below the surface, and then steeply decreased until it was below the detection limit at 4 mm. Zeaxanthin, an indicator for cyanobacteria, was detected in the upper 4 mm of the mat (5.34 μg m−3), and it was not found in the deeper parts of the studied mat. Its concentration was similar in the layers, where it was detected (5.10 ± 0.70 μg m−3), except for the layer at 3 mm that showed the highest concentration (9.4 μg m−3), which also showed the highest Chl a concentration. This indicates that cyanobacteria were found in the upper 4 mm of the mat, and they were mainly concentrated in the layer at 3 mm from the surface. On the other hand, β-carotene concentration remained similar between the layers (27.1±5.4 μg m−3) except in the layer at 3 mm, where it was two times higher (∼ 59 μg m−3) than that in other layers.As expected, the concentration of other accessory and protective pigments  decrease−1. Directly below the mat surface, incident irradiance was slightly enhanced (105%) because of high light scattering; conversely, only 1% of the incident irradiance (relative to that at the surface) was measured at 1.7 mm. Interestingly, light profiles showed regions of sudden local light attenuation (at 0.6 and 1 mm) that may correspond to the spots of high photosynthetic activity.A small fraction of incident irradiance was back-reflected from the mat surface (1.2%), whereas the rest (98.8%) was absorbed in the euphotic zone of the mat . Light exponentially attenuated below 0.3 mm in the mat at attenuation coefficient 3.76 mm2 concentration maxima at incident irradiance 130 μmol photons m−2 s−1 emerged directly below the surface (100 μm). Oxygen concentration increased to slightly more than saturation in the overlaying water (240 μM versus 200 μM), indicating limited photosynthetic activity. This was evidenced by measuring rates of volumetric gross photosynthesis using light–dark shift method (2 m−3 s−1) at 200 μm, whereas the active euphotic zone extended over 500 μm.Oxygen concentration rapidly increased as incident irradiance increased . The peat method , which s−2 s−1), O2 profile expanded over deeper region in the euphotic zone, in addition, O2 concentration reached a maximum of 1056 μM . Furthermore, this not only increased the rates of gross photosynthesis but also the depth of euphotic zone (1.2 mm) and number of peaks of activity (n  =  3). The rate in the first peak, which was directly below the surface, pronouncedly increased to 12.5 mmol O2 m−3 s−1 . Below the peak of SR activity, SR rates decreased progressively until 7 cm, where it slightly increased again (35.8 versus ∼ 20 nmol cm−3 d−1 in the upper layer). In the same layer, pronounced increase of sulfide emergence appeared after being close to zero in the upper 2 cm. In the deeper layers, both sulfate and sulfide progressively increased, without any considerable effect from SR, suggesting a deeper feeding source(s) of sulfate and sulfide.The integrated areal rates of sulfate reduction over a depth of 10 cm of the mat and those of the sediment underneath reached to 1558.4±426.2 nmol cmIn total, we investigated 8360 bacterial-, 2915 archaeal-, and 1484 eukaryotic-aligned sequences . From whDiversity and abundance of different members of the main phyla in the bacterial domain showed clear variations with depth . ProteobIn-depth analysis of the major phyla of domain Bacteria also showed variation in the sequence frequencies between different classes, as well as variations in the vertical distribution within members of the same class. When considering Proteobacteria , membersChroogleocystis and Chroococcus together, and Cyanothece that appeared only in L3 and L4. Contrarily, Mircocystis and SM1D11 were only found at the uppermost layer. Microcoleus sp. was detected starting from L2 and it had the highest sequence frequency in L4. Subsection III, Leptolyngbya sp., and Phoramidium sp. not only had the highest sequence frequencies at the mat surface, but also were the most diverse, whereas their presence and diversity in the deeper layers were much less.Overall, members of subsection III had the highest sequence frequency and were the most diverse cyanobacteria in Um Alhool mat. All cyanobacteria were more dominant in the uppermost layer compared with deeper layers  except fChloroflexi had generally more sequence frequency in the two deepest layers (L3 and L4), as conditions were more suitable for them than in the upper layers. Members of family Anaerolineaceae were the most abundant between all detected members of phylum Chloroflexi in the mat .2.1. Archaeal community analysis: Members of phylum Crenachaeota dominated the whole Archaeal domain in all layers of the studied mat (∼ 92%–97%) compared with members of Euryarchaeota (∼ 3–8%) . Overall2.2 Eukaryotic community analysis: In addition to bacterial and archael community analysis, we investigated the eukaryotic community structure in Um Alhool mat . The matUm Alhool mat is a very interesting self-sustaining microbial mat. It is an active and highly dynamic ecosystem that harbors a diverse array of microorganisms interacting with each other as well as with their environment. In spite of no surface sea water supply, the mat ecosystem is permanently covered with saline water in winter that seeps from below, which comes from adjacent Gulf water, in addition to limited freshwater precipitations during winter season (55.8 mm in 2011). Water chemistry analysis  demonstrOther nutrient resources such as dust deposition, river discharges, and nitrogen fixation by cyanobacteria cannot be ignored. The studied mat is located in an area that it is away from any human or industrial activities. Additionally, there are no rivers in the region that might provide nutrient-rich water to supply the mat system that is permanently surrounded by sand dunes. Therefore, nutrient content of the mat is, most probably, affected finitely by surrounding nutrients supply such as dust storms that occur in the summer season. The measurements in the current study were performed in winter; subsequently, the dust contribution to the nutrients in the mat was not determined. Nitrogen fixation by cyanobacteria is an important N-source for the microbial mat; however, its exact contribution was not determined in this study.a content. Indeed, the hyperspectral imaging of the Chl a signal (MPBI) in the upper layer of the mat  could be because of the inactive light absorption by abiotic components of the mat The mat surface reflected some incident irradiances, whereas the majority (∼ 98.8%) was absorbed in the ecosystem by photopigments see , detritu the mat  demonstra, phycocyanin, and carotenoids. These pigments characterize the oxygenic photosynthetic microorganisms such as cyanobacteria, diatoms, and eukaryotic algae, which showed elevated sequence frequencies in the upper layer compared with the deeper ones  based on their availability and the energy gain. The microbes, where O2 is available perform oxygenic respiration and in turn they gain the highest amount of energy. When O2 is consumed, nitrate respiration will start contributing more, followed by iron and manganese reduction. Considerable contribution to the organic materials oxidation in the microbial mats would come from sulfate reduction. In the deeper layers of the mat, the fermenters flourish and can be traced by their fermentation products such as organic acids and hydrogen. These products will serve as a suitable substrate for the methanogeic Archaea that contribute to the green-house gas emission.Ideally in microbial mats, photosynthetic microbes that consistently dominate the upper 1–2 mm, where light penetrates, are the main source of energy, organic material, and O−2 s−1). Microorganisms in the mats exposed to such extreme environmental conditions are equipped with different protective mechanisms to adapt to this environment. They can tune their photopigment production and distribution; also they migrate away to deeper layers where they can utilize the available light to the highest possible efficiency.Our results showed similar scenario, as the photoautotrophic communities  of the mat showed high photosynthetic rates, similar to those of other microbial mats (for example: −2 s−1), and is 1.5-times more than that of the upper 2 cm. The presence of a diverse eukaryotic community in the mat, particularly nematodes,  and sulfur-  reducing bacteria The oxygenic layers of the community were expected to be dominated by Cyanobacteria of the bacteria , which ibacteria , as the bacteria  to captuBacteria. Detected groups included organisms affiliated with the Methanosarcinales, the Halobacteriales, and uncultured groups of Euryarchaeota. In the studied mat, members belonging to Halobacterium were the most abundant and more diverse because they can tune their metabolism in response to changing in the environmental conditions. For example, in addition to their optimal growth under oxic conditions, they are capable for evolving a synchronized set of responses to adapt to oxygen limitation Members of domain Archaea had less sequence frequency, had lower diversity, and their distribution was less stratified than in Our results showed that the prokaryotic diversity exceeded that for Eukaryotes; a case that was also found in several other studies e.g., . This isFile S1SI file that contains detailed methodology describing sequence analysis, in addition to Table S1, Areal rates of photosynthesis in microbial mats, exposed to different incident intensities and temperatures. Figure S1, Rates of areal gross (red circle), net (black square) photosynthesis, and rates of respiration (blue triangles), at different incident irradiances.(DOCX)Click here for additional data file.


Food allergy is increasingly common worldwide. Tools for allergy diagnosis measuring IgE improved much since allergenic molecules and microarrays started to be used. IgE response toward allergens belonging to the same group of molecules has not been comprehensively explored using such approach yet.Using the model of lipid transfer proteins (LTPs) from plants as allergens, including two new structures, we sought to define how heterogeneous is the behavior of homologous proteins.Actinidia deliciosa) and gold (Actinidia chinensis) kiwifruit (KF), respectively, using clinically characterized allergic patients, and their biochemical features comparatively evaluated by means of amino acid sequence alignments. Along with other five LTPs from peach, mulberry, hazelnut, peanut, mugwort, KF LTPs, preliminary tested positive for IgE, have been immobilized on a microarray, used for IgE testing 1,003 allergic subjects. Comparative analysis has been carried out.Two new allergenic LTPs, Act d 10 and Act c 10, have been identified in green (Alignment of Act d 10 primary structure with the other allergenic LTPs shows amino acid identities to be in a narrow range between 40 and 55%, with a number of substitutions making the sequences quite different from each other. Although peach LTP dominates the IgE immune response in terms of prevalence, epitope recognition driven by sequence heterogeneity has been recorded to be distributed in a wide range of behaviors. KF LTPs IgE positive results were obtained in a patient subset IgE positive for the peach LTP. Anyhow, the negative results on homologous molecules allowed us to reintroduce KF in patients' diet.The biochemical nature of allergenic molecule belonging to a group of homologous ones should not be taken as proof of immunological recognition as well. The availability of panels of homologous molecules to be tested using microarrays is valuable to address the therapeutic intervention. Such claim seems to fit perfectly in the allergy field where the clinical phenotype is tightly linked to the IgE immune recognition and each patient seems to display a different clinical picture when compared to another. Such personalized approach, formerly almost impossible because of the number of tests to be performed in each patient, is now becoming increasingly feasible because of the biotechnology/microtechnology combination supported by information technology tools recently made available for the routine work Plant LTPs are widely distributed, structurally related, small proteins involved in defense mechanisms. Although their lipid-binding ability has been well reported, the biological function of LTPs is still largely unknown. The plant LTP family includes two subfamilies according to their molecular masses: the 9-kDa LTP1 and the 7-kDa LTP2. Although LTP1 and LTP2 share a common compact fold consisting of four α-helices stabilized by four disulfide bridges, the pairing partners of cysteines are not completely conserved between the two subfamilies, that also display a low overall sequence similarity (about 30% identity) www.allergome.org, accessed September 12, 2011).Up to now 63 LTPs have been characterized as allergens, being 46 of them expressed in edible parts of plants, almost all of them belonging to the LTP1 protein subfamily, and just two, having very preliminary reported data, to the LTP2 subfamily , we sought to compare the LTP biochemical, immunochemical, and clinical features in order to define the extent of their heterogeneity. The study, leading first to the full characterization of KF LTPs, brought us to abandon the former interpretation of “one molecule fits all” and to search for as much differences as possible among allergenic proteins facing each allergic patient, in order to increase the quality of personal decision making he or she deserves.The study received the approval of the Institutional ethical committee of the Istituto Dermopatico dell'Immacolata, Rome, Italy (66/CE/2006). Patients or caregivers signed an informed consent when undergoing tests not in the routine workup. Patients' demographical and clinical data, namely respiratory and ingestion-related symptoms on KF exposure, as well as all the in vivo and in vitro diagnostic data, were recorded for all patients by an allergy specialist, or transferred real time from the laboratory, into the InterAll software, a customized allergy electronic record for diagnostic and clinical data storing . KF specific clinical information were collected using the standard questionnaire reported in a previous study A subset of 259 consecutive subjects, tested nPru p 3 positive on the Immunosolid phase Allergen Chip (ISAC) 103 , was tested on the ISAC Exp96 . To verify the presence of LTP IgE positive subjects beside the nPru p 3 in the subset reported above, a control population of 744 consecutive allergic subjects tested positive to any allergen on ISAC 103 has been created. Overall 1,003 subjects underwent IgE testing with the ISAC Exp96 microarray.ST were performed and recorded as wheal areas using a standard methodology as already reported KF and peach DBPCFC have been performed as reported in literature in enrolled subjects when they did not report a recent anaphylaxis episode Total and specific IgE using a singleplex system were determined in patients' sera from venous blood samples obtained at the time of enrolment by the ImmunoCAP system . To verify the IgE binding capability of the new KF LTPs, biotinylated nAct d 10 and nAct c 10 (see below) have been coupled on a streptavidin-CAP following the standard methodology as suggested by the manufacturer (Phadia) and reported in literature www.allergome.org) Specific IgE for allergenic molecules were detected by ISAC 103 microarray test (PMD); ISAC tests were performed as previously reported IgE inhibition experiments have been performed applying the SPHIAa as previously reported Actinidia chinensis) and green KF (Actinidia deliciosa) were purchased at a local market. KFs were peeled, homogenized in a household blender after addition of 1 M NaCl (1∶1 w/v) and stirred at 4°C for 2 h. After centrifugation at 12,500 x g for 60 min, the supernatant representing the total protein extract was collected, dialyzed against water, aliquoted and stored at −20°C until used.Gold KF  and stirred at 4°C for 2 h. Seeds were crushed by pestle in a mortar until a smooth powder was obtained. Seed proteins were extracted in 0.5 M NaCl, at 4°C for 2 h. After protein extraction, pulp and seed samples were centrifuged at 12500 x The protein concentration in the extracts was estimated by the BIO-RAD Protein Assay , using calibration curves made with bovine serum albumin.RP- HPLC of KF extracts was performed on a Vydac  C8 column (0.21×25 cm), using a Beckman System Gold apparatus . Elution was accomplished by a multistep linear gradient of eluant B (0.08% TFA in acetonitrile) in eluant A (0.1% TFA) at a flow rate of 1 ml/min. The eluate was monitored at 220 and 280 nm. The separated fractions were manually collected and analyzed.KF LTPs, namely nAct c 10 and nAct d 10, were extracted from seeds and purified from the seed extracts using the procedure described by Ciardiello et al. The molecular mass of purified proteins was estimated by MALDI-TOF mass spectrometry measurements carried out on a PerSeptive Biosystems  Voyager-DE Biospectrometry Workstation. Analyses were performed on pre-mixed solutions prepared by diluting samples  in 4 volumes of matrix, namely 10 mg/ml α-ciano-4-hydroxycinnamic acid in 60% acetonitrile containing 0.3% TFA.In vitro simulated gastric digestion of nAct d 10 and nPru p 3 was performed as described by Bublin et al. www.expasy.org).Amino acid sequencing of LTP N-terminal region was performed with an Applied Biosystems Procise 492 automatic sequencer . Denaturation and alkylation of LTP sulfhydryl groups with 4-vinylpyridine was carried out as already described Purified nAct c 10 and nAct d 10 preparations  were incubated with 5 times molar excess of biotin N-hydroxysuccinimide ester  dissolved in DMSO (Sigma-Aldrich). After 3.5 hours at room temperature, the excess reagents were removed by gel-filtration chromatography using a PD10 column  equilibrated in PBS. The biotinylated preparations were then used for coupling with the streptavidin-CAP as described above.-µm filter , in a sterile horizontal laminar flow hood.Dialyzed protein extracts and purified LTP samples in deionized water were mixed with sterile glycerin in a 1∶1 ratio. The final protein concentration was 0.5 mg/ml. The LTP solutions were sterilized by membrane filtration through a 0.22e-record and underwent descriptive statistics including prevalence and median value for quantitative variables. Prevalence comparisons have been carried out using the χ2 test followed by pair-wise comparison using the Tukey post hoc test. Significances in inter-group value correlation comparisons were evaluated by the Spearman test. Specific IgE and ST results were expressed using median value, with interquartile ranges (5th percentile-95th percentile) (IQR). Serum IgE values were analyzed using the Kruskal-Wallis One-Way ANOVA by Ranks and the pair-wise comparison with a Mann-Whitney test with Bonferroni correction. Serum IgE percent value distribution obtained after the inhibition assay were compared using the Kruskal-Wallis One-Way ANOVA by Ranks, followed by the post hoc test Nemenyi-Damico-Wolfe-Dunn. The χ2 or Fisher's exact tests were applied to contingency tables and used when appropriate depending on the number of observations. All tests were used with two-sided options and significance level was set at a p value<0.05. Statistical analysis and graphical visualization of data have been performed using the R software (www.r-project.org) and Graphpad Prism .All diagnostic and experimental data were stored into the InterAll Seed and pulp extracts of green and gold KF were prepared, fractionated by RP-HPLC and the separated protein components were manually collected and analyzed by N-terminal amino acid sequencing. The analysis of a peak eluted at a retention time very similar to that observed for nPru p 3 allowed the identification of the LTP, namely Act c 10 and Act d 10, in the seed extracts from the gold and the green KFs, respectively, rather than from the pulp (see below). Act c 10 was eluted as a single peak, whereas Act d 10 was eluted in three overlapping peaks suggesting the presence of some isoforms. Direct protein sequencing of the purified protein allowed the identification of two different isoforms of Act d 10 (see below). Possible additional isoforms were not identified probably because of the low yield. Conversely, the LTP peak was absent in the RP-HPLC profiles obtained for the pulp extracts of the two KF species.−1 cm−1). Purity of the protein preparations was checked by SDS-PAGE, RP- HPLC and N-terminal amino acid sequencing.The recovery of the pure protein was about 0.4 mg per gram of green and gold KF seeds. The pulp was not used for LTP preparation. Protein concentration was estimated on the basis of the molar extinction coefficient at 280 nm , and encoded 5735 and 5737, respectively, in the Allergome database .The direct sequencing of the N-terminal region of native nAct d 10 produced 17 identifiable amino acid residues, AVSCGQVDTALTPCLTY . A smallThe analyses by MALDI-TOF mass spectrometry of purified nAct d 10 provided two values, 9.464 (±20) and 9.484 (±20) kDa, in good agreement with the mass values deduced from the amino acid sequence of the two isoforms having alanine (9.458 kDa) or treonine (9.488 kDa) as N-terminal residues, respectively. A molecular mass of 9.460 (±20) kDa was obtained for Act c 10 by mass spectrometry.The analysis by SDS-PAGE and RP-HPLC of the LTP samples subjected to digestion in SGF showed that nAct c 10 and nAct d 10, similarly to nPru p 3, are resistant to gastric digestion. Moreover, in line with the results reported by Cavatorta et al. www.expasy.org) showed the sequence identity between Act d 10 and other already known allergenic LTPs to be not very high, ranging between 55% with Ara h 9 (isoform Ara h 9.0201) and 35% with Par j 2. Identities among the full length amino acid sequence of the six allergenic LTPs under study  are comprised in the 42–70% range  confirming the positive IgE result on ISAC 103 used for selection, and replicated on ISAC Exp96 , without statistically significant differences when compared to ISAC 103 results. A slightly not statistically significant different skin reactivity was recorded when considering nAct c 10  and nAct d 10   . To set 2.6–159) , panel A2.6–159) , panel B2.6–159) , panel COverall the findings reported above preliminary marked immunological differences between the LTPs from peach and KFs, thus suggesting that the shared LTP reactivity recorded in the first set of patients and leading to the identification of KF LTPs could not be extended to all nPru p 3 positive subjects.As from the previous section findings, showing heterogeneity between LTPs from two allergenic sources in that small study group, a larger study was set using the routine testing approach by the ISAC microarray, in order to define any possible subsets related to different LTP IgE recognition. A comparative evaluation of seven LTPs was undertaken using the ISAC 103 and ISAC Exp96 microarrays in parallel.versus KF LTPs, peanut and mulberry ones. Absence of nAct d 10 exclusively IgE positive subjects statistically differed to all the remaining LTPs rather than nAct c 10 (data not shown). Raw prevalence are reported in 2 test has been obtained taking together the seven prevalence, whereas not all prevalence were statistically different when compared each other. For instance, nPru p 3 was anyway the most prevalent sensitization with no difference only when compared to nMor n 3. IgE sensitization to nAct c 10 and nAct d 10 had statistically significant higher prevalence than the mugwort and the hazelnut LTPs, but less prevalent than nMor n 3 and nPru p 3, though proven by statistics just for nPru p 3. The less prevalent IgE reactivity have been recorded for the mugwort and the hazelnut LTPs being statistically different from all other LTPs. Detailed statistics are reported in Unless the original two population were defined on the basis of being nPru p 3 IgE positive or negative as tested on ISAC 103, additional nPru p 3 positive subjects were found in the control population using the ISAC Exp96 microarray bearing a different nPru p 3 preparation, thus raising the number of nPru p 3 positive subjects from 259 to 296. The same control population revealed a number of subjects being nPru p 3 IgE negative but positive to at least one of the other LTPs, increasing the overall number of subjects being positive to at least one of the LTPs from 296 to 431. nAct c 10, nAct d 10, rAra h 9, nArt v 3, rCor a 8, and nMor n 3 were positive when nPru p 3 was negative in 48, 41, 17, 56, 43, and 17 cases, respectively. The number of subjects positive to one LTP as listed above and negative to all others was relatively low, being 5, 0, 9, 25, 10, 9, 17. These prevalence were statistically different only when considering nArt v 3 values To visualize exclusive sensitization to one LTP compared to others, a series of Venn diagrams showing all possible logical relations between our finite collections of IgE values obtained by the two microarray testing, have been generated and reported in A cluster analysis performed using all subjects having IgE positive results to at least one of the seven LTPs generated the heat map shown in Beside the evaluation of positive/negative results reported above and clearly showing non-overlapping behavior among LTPs, we sought to verify whether detected IgE value distributions were different comparing the seven LTPs under study. A Kurskal-Wallis test gave statistically significant results (p<0.0001), and, when comparing the paired groups using the Mann-Whitney test several statistically different findings were recorded at different p values . Differiversus nAct d 10 correlation was taken, having the highest Spearman r (0.94) and χ2 (851.6) values  (2 (107.2) when comparing nAct c 10 and nArt v 3  values , panel A= 761.8) , panel VnArt v 3 , panel EnArt v 3 , panel OnArt v 3 , panel SnArt v 3 , panel SnArt v 3 , panel OTo define immunological relationships in terms of IgE binding among LTPs, the SPHIAa assay was run using nAct c 10, nAct d 10, and nPru p 3 as inhibitor on the seven LTPs. The assay was performed using ten individual sera selected for being IgE positive to the three inhibitors as shown in To verify whether the heterogeneous immunological behavior between the two KF LTPs and the peach LTP could lead to define a subset of patients who are also not clinically reactive to all LTPs, we first performed a series of experiments to define the LTP distribution within the KF tissues, namely the pulp and the seeds, based on biochemical, immunochemical and clinical methods.in vivo skin testing with green KF seed and pulp preparations in 21 selected subjects. As reported in As reported in the first paragraph of the in vivo clinical reactivity to green KF in well characterized peach allergic patients. We thus recruited patients having different diagnostic profiles. All enrolled ones had to be nPru p 3 positive with either a positive DBPCFC to peach or a recent severe generalized reaction on peach ingestion. All underwent the tests as reported in in vivo or in in vitro tests, whereas among the KF tolerant in the majority of the cases the KFs LTP were tested with negative results, but with exceptions, unless positive values were in the lowest range for both ISAC and ST .We herein report for the first time the identification of two new LTPs from the gold and the green KFs and their characterization as clinically relevant allergens. The primary structure of nAct d 10, the one from green KF, has been fully elucidated, being the one of nAct c 10 from gold KF very similar, mainly on the basis of the almost overlapping findings described throughout the present study. The identification process was straight forward as, using a panel of other KF allergens we selected patients being IgE negative for them and having just a positive IgE result for a potentially homologous molecule, namely nPru p 3. This process is highly suggested as leads to a secured evaluation of the newly identified structure, without overlapping positivity as sometimes reported in literature Unless we started the identification using subjects having the double clinical reactivity to peach and KF, the first evidence that we were dealing with new allergens not being part of a tight homogeneous group of molecules came by the comparative evaluation of primary structures and the preliminary extended parallel comparative testing with the peach LTP on the 44 peach allergic subjects. The former is the biochemical evidence that Act d 10 is not closely related to anyone of the already known allergenic LTPs. In fact, the observed sequence identities are never higher than 55% and generally fall in the narrow range of 40–55%, whereas more closely related LTPs display higher values. For instance, identities of 97, 91, 88, 87 and 79% are obtained when Pru p 3 sequence is compared with Pru du 3, Pru ar 3, Pru d 3, Pru av 3, Mal d 3 ones, respectively About comparative testing, our data on a huge population where just one LTP, nPru p 3, was tested did not arise such suspicion Regarding molecule to molecule immunological relationships, some studies reported on the need of studying conformational epitopes shared or not by different LTPs In the need of profiling patients reporting KF allergy, we used in collaboration with other research groups, Pru p 3 as representative LTP also for KF LTP in vivo/in vitro results as herein reported.Overall we can describe the clinical relevance of our findings as follows: we found non-symptomatic subjects as in previous studies After all, it remains that LTP represent a cross-sectional group of allergens having a patient's related IgE co-recognition. What should be avoided is the interpretation of LTP as part of a given allergenic source in the so called component-resolved diagnosis Last important finding of our study is the original distribution of LTP within the KF tissues. LTP is generally reported to be found on the surface of fruits, as in the case of peach In conclusion, we suggest the use of the microarray testing system bearing panels of homologous molecules as it adds much to our comparative knowledge on biochemical, immunochemical and clinical differences between closely related structures. Clinical allergists might take advantage of missing IgE recognition of one or the other LTP for keeping the specific food in patient's diet, avoiding useless exclusion, generally leading to deterioration of patient's quality of life.Furthermore, we herein document the need of having the microarray-based testing available for routine allergy diagnosis workup, as it creates the conditions for generating a wealth of data without selection biases and on cohorts otherwise not approachable, as we cannot exclude a different behavior of patients living in different geographical areas. As preliminary depicted in a review by us