Age- and ethnicity-related reference intervals for serum vitamin B12.

BACKGROUND: Age and ethnicity are known to influence serum vitamin B12 (B12) concentration, yet universal reference intervals (RIs) are typically applied by laboratories. Both lower and upper RI limits for B12 are clinically relevant. Low values suggest deficiency leading to anemia and/or neurological impairment, while high values are not always an innocuous consequence of high B12 intake but are associated with some cancers, autoimmune, liver, and renal diseases. This work aimed to establish age- and ethnicity-related RIs for B12 using a modified indirect method based on Hoffmann's approach. METHODS: A total of 72,091 anonymized B12 results (Jan 2018-Nov 2019) were analyzed from an ethnically-diverse South-East London general practice patient population. Patients belonged to five ethnic groups: Asian, Black, White, Mixed, or Other. Multiple records for the same patient and results with missing ethnicity were excluded from the analysis of adult RIs. B12 analyses were performed using ARCHITECT® (Abbott Diagnostics). RESULTS: B12 was significantly higher in Black compared with Asian and White adults. There were no differences in B12 between Asian and White adults. Children (all ethnicities) between 2 and 5 years old had the highest B12. Because of the small number of children (up to the age of 13) in each ethnic-related age category, all ethnic groups were combined to obtain age-related RIs. The children's RIs ranged from 159 to 1025 pmol/L for 0-1-year-olds to 276-1102 pmol/L for 2-5-year-olds. The RIs for Black and White/Asian people >13 years of age were 166-805 pmol/L and 134-511 pmol/L respectively. CONCLUSIONS: The application of age- and ethnicity-appropriate RIs into diagnostic practice will provide a more accurate evaluation of B12 status when using the B12 test alone or in combination with other markers.

The administration of amnion-derived multipotent cell secretome ST266 protects against necrotizing enterocolitis in mice and piglets.

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants and is steadily rising in frequency. Patients who develop NEC have a very high mortality, illustrating the importance of developing novel prevention or treatment approaches. We and others have shown that NEC arises in part from exaggerated signaling via the bacterial receptor, Toll-like receptor 4 (TLR4) on the intestinal epithelium, leading to widespread intestinal inflammation and intestinal ischemia. Strategies that limit the extent of TLR4 signaling, including the administration of amniotic fluid, can reduce NEC development in mouse and piglet models. We now seek to test the hypothesis that a secretome derived from amnion-derived cells can prevent or treat NEC in preclinical models of this disease via a process involving TLR4 inhibition. In support of this hypothesis, we show that the administration of this secretome, named ST266, to mice or piglets can prevent and treat experimental NEC. The protective effects of ST266 occurred in the presence of marked TLR4 inhibition in the intestinal epithelium of cultured epithelial cells, intestinal organoids, and human intestinal samples ex vivo, independent of epidermal growth factor. Strikingly, RNA-seq analysis of the intestinal epithelium in mice reveals that the ST266 upregulates critical genes associated with gut remodeling, intestinal immunity, gut differentiation. and energy metabolism. These findings show that the amnion-derived secretome ST266 can prevent and treat NEC, suggesting the possibility of novel therapeutic approaches for patients with this devastating disease.NEW & NOTEWORTHY This work provides hope for children who develop NEC, a devastating disease of premature infants that is often fatal, by revealing that the secreted product of amniotic progenitor cells (called ST266) can prevent or treat NEC in mice, piglet, and "NEC-in-a-dish" models of this disease. Mechanistically, ST266 prevented bacterial signaling, and a detailed transcriptomic analysis revealed effects on gut differentiation, immunity, and metabolism. Thus, an amniotic secretome may offer novel approaches for NEC.

Exploratory Phase II Multicenter, Open-Label, Clinical Trial of ST266, a Novel Secretome for Treatment of Persistent Corneal Epithelial Defects.

Objective: An exploratory phase II, multicenter, open-label, clinical trial (NCT03687632) was conducted to evaluate the safety and effectiveness in treating persistent corneal epithelial defects (PEDs) with ST266, a proprietary novel multi-cytokine platform biologic solution secreted by cultured Amnion-derived Multipotent Progenitor (AMP) cells. Methods: Subjects with a PED were treated with ST266 eye drops 4 times daily for 28 days, then followed for 1 week. Safety was assessed by monitoring of adverse events (AEs) and serious adverse events (SAEs). Efficacy was assessed by measuring the area of the PED by slit lamp biomicroscopy. Tolerability of ST266, percentage of eyes with complete healing, reduction in area of the epithelial defect, and maintenance of a reduction in the area of the epithelial defect 7 days after treatment were recorded. Results: Thirteen patients were enrolled into the trial at one of eight sites. The first patient withdrew after 5 days. The remaining 12 patients with PEDs with median duration of 39 days (range = 12 to 393 days) completed treatment. Ten of the 12 eyes had been refractory to treatment with various conventional therapies prior to enrollment. After 28 days of treatment, there was a significant decrease in mean PED area compared with baseline (66.4% ± 35.3%, P = 0.001). At follow-up, 1 week after completion of treatment, on day 35, the PED area was further reduced by 78.8% ± 37.5% (P = 0.01) compared with baseline. During 28 days of treatment, 5 eyes (41.7%) had complete wound closure. There were no AEs of concern thought to be related to the drug, and no SAEs were noted. Conclusions: In this trial, we found ST266 eye drops might promote corneal epithelization, thereby reducing the PED area, including in refractory cases in a wide range of etiologies. ST266 was well-tolerated by most patients.

Ethnicity influences total serum vitamin B12 concentration: a study of Black, Asian and White patients in a primary care setting.

AIMS: A growing body of evidence suggests that ethnicity and race influence vitamin B12 metabolism and status yet clinical awareness of this is poor, causing doubts regarding diagnosis and treatment. Moreover, deficiency and insufficiency cut-offs are universally applied for this test in most diagnostic settings. The objective of this study was to assess serum vitamin B12 concentrations in Black, Asian and White primary care patients in London, UK, particularly in patients of Black or Black British ethnic origin and establish if there is a need for specific reference ranges. METHODS: Serum B12 results from 49 414 patients were processed between January 2018 and November 2019 using the Architect assay (Abbott Diagnostics) at St. Thomas' Hospital, London, UK. Age, sex and ethnicity data were collected from the laboratory Health Informatics Team. RESULTS: Black patients (n=13 806) were found to have significantly higher serum vitamin B12 concentration across all age groups and both sexes, especially Nigerian patients (median B12 505 pmol/L,IQR: 362-727, n=891), compared with Asian and White ethnic groups (p<0.001). Binary logistic regression analysis revealed that the Black or Black British ethnic group had the strongest association with elevated serum B12 (>652 pmol/L) (adjusted OR 3.38, 95% CI 3.17 to 3.61, p<0.0001). CONCLUSIONS: It is likely that a combination of genetic and acquired/environmental factors are responsible for the ethnic differences in serum B12. This suggests that there is a need for ethnic-specific reference ranges with indications for the incorporation of age and sex too.

Use of amnion-derived cellular cytokine solution for the treatment of gingivitis: A 2-week safety, dose-ranging, proof-of-principle randomized trial.

BACKGROUND: A 6-week Phase I clinical trial was performed to primarily evaluate the safety and secondarily determine the preliminary efficacy of a novel biological solution, ST266, comprised of a mixture of cytokines, growth factors, nucleic acids, and lipids secreted by cultured amnion-derived multipotent progenitor cells on gingival inflammation. METHODS: Fifty-four adults with gingivitis/periodontitis were randomly assigned to 1X ST266 or diluted 0.3X ST266 or saline topically applied on facial/lingual gingiva (20 µL/tooth). Safety was assessed through oral soft/hard tissue exam, adverse events, and routine laboratory tests. Efficacy was assessed by modified gingival index (MGI), bleeding on probing, plaque index, probing depth (PD), and clinical attachment level (CAL). Assessments were performed on day 0, 8, 12, and 42. ST266 and saline applied daily starting at day 0 through day 12 except weekend days. Plasma was analyzed for safety and proinflammatory cytokines, interleukin (IL)-1ß, IL-6, tumor necrosis factor-alpha, and interferon gamma. Gingival crevicular fluid (GCF) was analyzed for the same cytokines. Subgingival plaque was primarily analyzed by checkerboard DNA-DNA hybridization. Comparisons with saline were modeled through a generalized estimating equations method adjusting for baseline. RESULTS: No safety concern was found related to ST266. Statistically significant reduction in MGI was noted at day 42 by 1X ST266 compared with saline (P = 0.044). PD and CAL were reduced by both doses of ST266 at day 42 (P <0.01) and by 1X ST266 at day 12 (P <0.05). GCF IL-1ß and IL-6 levels were reduced by both doses of ST266 at day 12 (P <0.05, P <0.01, respectively). IL-6 was also significantly reduced in plasma of both ST266 groups (P <0.05). Significant reductions in red complex bacteria were detected in both ST266 doses. CONCLUSIONS: In this "first in human oral cavity" study, topical ST266 was safe and effective in reducing gingival inflammation in 6 weeks. Longitudinal studies with large sample sizes are warranted to assess the therapeutic value of this novel host modulatory compound in the treatment of periodontal diseases.

Safety Profile of Amnion-Derived Cellular Cytokine Solution (ACCS) Following Topical Skin Application in Patients Receiving Breast Radiotherapy.

OBJECTIVE: To establish a safety profile for amnion-derived cellular cytokine solution following topical application in patients undergoing whole breast radiotherapy for breast cancer. MATERIALS AND METHODS: Twenty female patients with early-stage breast cancer were enrolled in 2 separate cohorts of an institutional review board-approved phase I protocol. Cohort 1 consisted of 10 patients who received topical amnion-derived cellular cytokine solution to the breast immediately following the first 10 fractions of whole breast radiotherapy. Cohort 2 consisted of 10 additional patients who fit the same criteria as the initial cohort but received topical amnion-derived cellular cytokine solution following the development of at least grade I breast erythema. Blood samples were tested for the presence of proteins in amnion-derived cellular cytokine solution as well as routine hematologic functions. RESULTS: Amnion-derived cellular cytokine solution did not induce overproduction of any cytokines sampled, and there was no evidence of "cytokine storm." It also showed no significant absorption systemically following topical delivery. No patients developed an adverse event. There were no patterns of changes in vital signs or clinical laboratory tests that were related to the treatment regimen. CONCLUSION: In this safety trial, the topical application of amnion-derived cellular cytokine solution in both intact and denuded, irradiated skin was found to be safe, and showed no evidence of systemic absorption. No cosmetic changes were identified long term. Patient blood chemistry was not adversely affected, indicating the absence of an anaphylactic response and no evidence "cytokine storm" was identified. Amnion-derived cellular cytokine solution is safe to use topically in clinical protocols.

A simple, rapid, and convenient Luminex-compatible method of tissue isolation.

Various pathological conditions are associated with changes in multiple protein biomarkers, and these changes can be assessed using xMAP beads and the Luminex platform. Although this platform is most commonly utilized in the analysis of biomarkers in serum, plasma, or urine, it is often desirable to measure these analytes in tissues (e.g., biopsy specimens). We have developed a simple, rapid method of tissue isolation in which excised tissues are permeabilized in RNAlater at 4 degrees C overnight, followed by Luminex analysis for inflammatory biomarkers (cytokines). This method was compared with flash-freezing in both mouse liver and human debrided wound specimens, and may be of utility in other clinical settings.

The use of amnion-derived cellular cytokine solution to improve healing in acute and chronic wound models.

OBJECTIVE: Growth factors demonstrate mixed results improving wound healing. Amnion-derived multipotent cells release physiologic levels of growth factors and tissue inhibitors of metalloproteinases. This solution was tested in models of acute and chronic wound healing. METHODS: Acute model: Sprague-Dawley rats underwent laparotomy incisions. The midline fascia was primed with phosphate-buffered saline, unconditioned media, or amnion-derived cellular cytokine suspension prior to incision. Breaking strength of laparotomy wounds was tested with an Instron tensiometer. Incisional hernia formation was measured after 28 days. Chronic model: Chronic, infected granulating wounds were produced in rats by excising full thickness burn eschars inoculated with Escherica coli. Granulating wounds were treated with unconditioned media or amnion-derived cellular cytokine suspension. Treatments were applied either on day 0 and day 7 or day 0 and then every other day. Wounds were traced every 72 hours and biopsied for quantitative bacteriology. RESULTS: Acute model: Priming with amnion-derived cellular cytokine suspension increased the breaking strength of laparotomy incisions in comparison with phosphate-buffered saline or unconditioned media (P < .05). Acute wound failure and incisional hernia formation was 100% in the phosphate-buffered saline and unconditioned media groups and 18% in the amnion-derived cellular cytokine suspension-treated group (P <.05). Chronic model: The rate of wound closure was accelerated in amnion-derived cellular cytokine suspension-treated chronic wounds (P < .05). Multidosing improved the effect. CONCLUSIONS: A physiologic solution of cytokines and tissue inhibitors of metalloproteinases improves healing in models of acute and chronic wounds. Such a cocktail can be produced from amnion-derived multipotent progenitor cells.

Amnion-derived cellular cytokine solution: a physiological combination of cytokines for wound healing.

OBJECTIVE: Wound repair is a complex integration of dynamic processes mediated by humeral messages controlling the levels of cytokines, growth factors, and matrix metalloproteinases in the wound space. Isolated growth factors and growth factor combinations have been used to accelerate wound healing with limited success. A cellular cytokine solution can be collected by harvesting the proteins released from amnion-derived multipotent progenitor cells. The purpose of this study was to compare levels of cytokines/growth factors in amnion-derived cellular cytokine solution with physiological levels reported in the medical literature. METHODS: Amnion-derived multipotent progenitor cells were grown to confluency, and the proteins secreted were characterized by qualitative and quantitative analysis. These results were compared with physiologic levels reported in the medical literature. RESULTS: The results demonstrated that amnion-derived cellular cytokine solution contained physiologic levels of cytokines relevant to wound healing, including platelet-derived growth factor, vascular endothelial growth factor, angiogenin, transforming growth factor beta 2, tissue inhibitor of metalloproteinase-1, and tissue inhibitor of metalloproteinase-2. The ranges (mean +/- standard deviation) were as follows: platelet-derived growth factor, 86 +/- 33 pg/mL; vascular endothelial growth factor, 5.7 +/- 1.5 ng/mL; angiogenin, 1.0 +/- 0.33 ng/mL; transforming growth factor beta 2, 500 +/- 330pg/mL; tissue inhibitor of metalloproteinase-1, 530 +/- 140 ng/mL; and tissue inhibitor of metalloproteinase-2230 +/- 40 ng/mL. These levels are comparable with the physiologic levels reported in the literature. CONCLUSIONS: The physiologic levels of cytokines important to healing found in amnion-derived cellular cytokine solution suggest that amnion-derived cellular cytokine solution may be of benefit in healing certain acute and chronic wounds.

Agent-based model of inflammation and wound healing: insights into diabetic foot ulcer pathology and the role of transforming growth factor-beta1.

Inflammation and wound healing are inextricably linked and complex processes, and are deranged in the setting of chronic, nonhealing diabetic foot ulcers (DFU). An ideal therapy for DFU should both suppress excessive inflammation while enhancing healing. We reasoned that biological simulation would clarify mechanisms and help refine therapeutic approaches to DFU. We developed an agent-based model (ABM) capable of reproducing qualitatively much of the literature data on skin wound healing, including changes in relevant cell populations (macrophages, neutrophils, fibroblasts) and their key effector cytokines (tumor necrosis factor-alpha [TNF], interleukin [IL]-1beta, IL-10, and transforming growth factor [TGF]-beta1). In this simulation, a normal healing response results in tissue damage that first increases (due to wound-induced inflammation) and then decreases as the collagen levels increase. Studies by others suggest that diabetes and DFU are characterized by elevated TNF and reduced TGF-beta1, although which of these changes is a cause and which one is an effect is unclear. Accordingly, we simulated the genesis of DFU in two ways, either by (1) increasing the rate of TNF production fourfold or (2) by decreasing the rate of TGF-beta1 production 67% based on prior literature. Both manipulations resulted in increased inflammation (elevated neutrophils, TNF, and tissue damage) and delayed healing (reduced TGF-beta1 and collagen). Our ABM reproduced the therapeutic effect of platelet-derived growth factor/platelet releasate treatment as well as DFU debridement. We next simulated the expected effect of administering (1) a neutralizing anti-TNF antibody, (2) an agent that would increase the activation of endogenous latent TGF-beta1, or (3) latent TGF-beta1 (which has a longer half-life than active TGF-beta1), and found that these therapies would have similar effects regardless of the initial assumption of the derangement that underlies DFU (elevated TNF vs. reduced TGF-beta1). In silico methods may elucidate mechanisms of and suggest therapies for aberrant skin healing.

Clinical evaluation of recombinant human platelet-derived growth factor for the treatment of lower extremity ulcers.

BACKGROUND: Diabetic foot ulcer is a major health care problem that leads to amputation. METHODS: Patients with full-thickness diabetic neurotrophic foot ulcers present for longer than 8 weeks without healing were entered into one of five randomized, prospective, blinded clinical trials comparing treatment with platelet-derived growth factor (PDGF) or placebo gel for up to 20 weeks. The purpose of these trials was to assess the safety and efficacy of PDGF used with good wound care. Each patient had an adequate arterial blood supply, was free of infection, was off-loaded, and was extensively débrided. The ulcers had been present for at least 8 weeks. RESULTS: A total of 922 patients were entered into the study. Analysis of ulcers with a baseline area of less than 10 cm (95 percent of patients) showed that patients treated with PDGF at 100 mug/g had a significant increase in complete healing compared with patients given placebo (50 percent versus 36 percent, p < 0.007). PDGF also decreased the time to complete healing by 30 percent (14 weeks versus 20 weeks, p = 0.01). Adverse events were similar in both treatment groups, as were recurrent ulcer rates. CONCLUSION: PDGF applied once daily was effective in healing chronic diabetic neurotrophic foot ulcers when used in conjunction with good wound care.

Wound-healing trajectories as outcome measures of venous stasis ulcer treatment.

Outcome measures of venous ulcer healing are not uniformly accepted. Stringent criteria of 100% closure fail to provide information of healing over the entire span of repair. Wound-healing trajectories (plot of percentage of wound closure versus time of wound treatment) were constructed for 232 patients treated in eight clinical trials at two independent wound care/research centres. Trajectories were constructed for ulcers that totally healed (100% closure) and those that did not (<100% closure) over a 20-week period. Kaplan-Meier survival plots determined the percentage of patients achieving total healing versus time of treatment. The wound-healing trajectories were almost identical for patients achieving complete ulcer healing, as were the trajectories for patients with less than 100% closure. The trajectories for the ulcers healing completely were significantly different from those with <100% closure. Only 60% of all patients achieved 100% closure by 20 weeks. Using linear regression, it was predicted that it would take 31 weeks for all patients to achieve total healing. Total healing is an inadequate outcome measure for healing of venous stasis ulcers. Clinical trials using this measure would require excessive time periods. As wound-healing trajectories for patients treated at two centres mimic one another, shifting the wound-healing trajectory from one of impaired healing to one of a more ideal healing course may be considered a better outcome measure for determining healing of venous stasis ulcers.

Debridement.

Debridement is defined as the removal of nonviable material, foreign bodies, and poorly healing tissue from a wound. Although surgeons recognize the importance of debridement, few data have been generated in randomized trials to support its use. Debridement provides for removal of tissue with the highest bacterial count, reliable cultures, and identification of osteomyelitis. The most direct form of debridement is surgical excision. For patients who are poor candidates for surgical debridement or have limited access to a surgeon, other forms of debridement (including mechanical, autolytic, enzymatic, and biologic) can be used. Although operative debridement is best performed by those with surgical training, the other forms of debridement can be accomplished by other allied health care professionals. Debridement remains an important adjunct to good wound care, but questions of what type, how much, and how often it should be performed remain unresolved.

Wound-healing trajectories.

Healing of a wound is a dynamic process involving soluble mediators, a variety of cells, and extracellular matrix. These components are involved in a number of different processes or steps in healing, including coagulation, inflammation, fibroplasia, collagen deposition, epithelialization, and scar contraction with remodeling. The processes can be organized into three phases: inflammation, fibroplasia, and remodeling. The events in healing occur in an orderly and timely fashion, and there is significant overlap between each of the processes.