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BackgroundThe shipment of human islets (IE) from processing centers to distant laboratories is beneficial for both research and clinical applications. The maintenance of islet viability and function in transit is critically important. Gas-permeable silicone rubber membrane (SRM) vessels reduce the risk of hypoxia-induced death or dysfunction during high-density islet culture or shipment. SRM vessels may offer additional advantages: they are cost-effective (fewer flasks, less labor needed), safer (lower contamination risk), and simpler (culture vessel can also be used for shipment).MethodIE were isolated from two manufacturing centers and shipped in 10-cm(2) surface area SRM vessels in temperature- and pressure-controlled containers to a distant center after at least 2 days of culture (n = 6). Three conditions were examined: low density (LD), high density (HD), and a microcentrifuge tube negative control (NC). LD was designed to mimic the standard culture density for IE preparations (200 IE/cm(2)), while HD was designed to have a 20-fold higher tissue density, which would enable the culture of an entire human isolation in 1-3 vessels. Upon receipt, islets were assessed for viability (measured by oxygen consumption rate normalized to DNA content [OCR/DNA)]), quantity (measured by DNA), and, when possible, potency and function (measured by dynamic glucose-stimulated insulin secretion measurements and transplants in immunodeficient B6 Rag(+/-) mice). Postshipment OCR/DNA was not reduced in HD vs LD and was substantially reduced in the NC condition. HD islets exhibited normal function postshipment. Based on the data, we conclude that entire islet isolations (up to 400,000 IE) may be shipped using a single, larger SRM vessel with no negative effect on viability and ex vivo and in vivo function.

Background: Reducing antimicrobial resistance (AMR) requires a multidisciplinary One Health approach, which necessitates buy-in from all stakeholders. In Aotearoa New Zealand, where the dairy industry is one of the largest users of antimicrobials, there are ongoing efforts to optimise antimicrobial usage (AMU) to minimise the development of AMR. These include regulations around the veterinary authorisation of the use of antibiotics by farmers without the need for a specific prescription ("the RVM process") and programmes such as the New Zealand Veterinary Association's antibiotic 'Traffic Light System'. The goal of this pilot survey was to develop and trial a questionnaire to determine how much Aotearoa dairy farmers understand about One Health, AMR, the RVM process and how their actions regarding AMU affect the wider environment., Methods: A 55-question semi-structured questionnaire was piloted on 15 dairy farms in the Lower North Island of Aotearoa New Zealand via an in-person semi-structured interview between September and November 2021., Results: None of the interviewed farmers could define the term One Health. However, the majority found the RVM process to be of use on their farm, although admitted they generally felt frustration regarding AMR, seeing it as a blockage to productivity, and lacked awareness regarding how their actions were related to its development. Of the farmers interviewed over half had not heard of the traffic light system, and of those who had, one admitted they refused to adhere to it., Conclusions: This survey's novel findings have highlighted that there are notable gaps within dairy farmer understanding of AMU, AMR and One Health as well as highlighting that veterinarians could do more to keep their clients informed of their important role within One Health. There is still a lot more work to do with regards to vets, farmers and industry representatives working together to embrace One Health. Simple solutions would be to encourage farmers returning unused drugs to their veterinarians for correct disposal and to actively engage farmers further regarding AMU and AMR, so that these end-product users do not feel disconnected from the process., (© 2024. The Author(s).)

Introduction: Conventional culture conditions, such as in T-flasks, require that oxygen diffuse through the medium to reach the islets; in turn, islet surface area density is limited by oxygen availability. To culture a typical clinical islet preparation may require more than 20 T-175 flasks at the standard surface area density of 200 IE/cm 2 . To circumvent this logistical constraint, we tested islets cultured on top of silicon gas-permeable (GP) membranes which place islets in close proximity to ambient oxygen., Methods: Oxygenation of individual islets under three culture conditions, standard low-density, non- GP high density, and GP high density, were first modeled with finite element simulations. Porcine islets from 30 preparations were cultured for 2 days in devices with GP membrane bottoms or in paired cultures under conventional conditions. Islets were seeded at high density (HD, ∼4000 IE/cm 2 , as measured by DNA) in both GP and non- GP devices., Results: In simulations, individual islets under standard culture conditions and high density cultures on GP membranes were both well oxygenated whereas non- GP high density cultured islets were anoxic. Similarly, compared to the non -GP paired controls, islet viability and recovery were significantly increased in HD GP cultures. The diabetes reversal rate in nude diabetic mice was similar for HD GP devices and standard cultures but was minimal with non -GP HD cultures., Discussion: Culturing islets in GP devices allows for a 20-fold increase of islet surface area density, greatly simplifying the culture process while maintaining islet viability and metabolism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Avgoustiniatos, Mueller, Scott III, Kitzmann, Suszynski, Perrault, Falde, Balamurugan, Hering, Putnam and Papas.)

Subjective pain assessment in cattle is contingent upon the observer's experience and attitude. Studies of pain assessment in cattle by veterinarians and farmers using different pain scales have been published. This systematic review and meta-analysis aimed to describe and compare the pain scores given by veterinarians and producers for different procedures and conditions using either a NRS or VAS. The literature search was conducted with PubMed (MEDLINE) and Agricola, using defined search terms (e.g., peer-reviewed). A total of 842 articles were identified. After screening of duplicates, abstracts, and full texts, a total of 16 articles were included in this systematic review. Different pain scales were used for the included studies (NRS 0-10 for eight studies, NRS 1-10 for six studies, NRS 1-10 and VAS 0-10 for one study, and VAS 0-1 for one study). Most studies ( n = 11) originated from the European Union. Mean values for pain scores differed significantly between studies included in the meta-analysis for both NRS 0-10 and 1-10. The findings of this study indicated that comparison of pain scoring used in different studies is difficult due to use of different pain scales and varying nomenclature, and that many variables (such as age and gender) influence pain scoring., Competing Interests: The authors declare no conflicts of interest.

Lameness detection is a significant challenge. Locomotion scoring (LS), the most widely used system for detecting lameness, has several limitations, including its subjective nature and the existence of multiple systems, each with its own advantages and disadvantages. Therefore, this study aimed to evaluate whether the foot skin temperature (FST) of hind limbs, as measured using infrared thermography (IRT), could potentially be used as an alternative on Tanzanian dairy farms. Each of the three study farms were visited twice during the afternoon milking on consecutive days, with a total of 170 cows assessed. DairyNZ LS (4-point scale (0-3)) was undertaken on the first day as the cows exited the milking parlour after being milked, while on the following day, the plantar aspect of the hind limbs of the cows was thermally imaged while they were standing in the milking parlour, using a handheld T650sc forward-looking infrared camera. Mean FST was higher for cows with a locomotion score of 1 than those with a score of 0; higher for cows with a locomotion score of 2 than those with a score of 1; and higher for cows with a locomotion score of 3 than those with a score of 2, with each one-unit locomotion score increase being associated with a 0.57 °C increase in mean temperature across all zones. The optimal cut-off point of 38.0 °C for mean temperature across all zones was identified using a receiver operator characteristic curve. This cut-off point had a sensitivity of 73.2% and a specificity of 86.0% for distinguishing cows with a locomotion score ≥ 2 (clinical lameness). The prevalence of clinical lameness across all three farms was 33%, which meant that only 72% of cows with a mean FST across all zones ≥ 38.0 °C had been identified as clinically lame using LS. This study confirmed that IRT has the potential to be used to detect lameness on Tanzanian dairy farms. However, before it can be widely used, improvements in accuracy, especially specificity, are needed, as are reductions in equipment (IR camera) costs.

One common management strategy used to reduce the risk of lameness is prophylactic claw trimming. However, in pasture-based cattle, there is a concern that the immediate reduction in sole thickness resulting from sole trimming will lead to medium-to-long-term reductions in sole thickness, which may increase the risk of lameness. Nevertheless, there is a lack of data on sole thickness and trimming in pasture-based cows. Therefore, the aim of this study was to evaluate the effect of trimming on sole thickness over the medium-to-long term, as estimated using the ultrasound-measured distance from the external claw sole surface to the distal phalanx (DDP) and of DDP on the interval between calving and increased locomotion scores. A total of 38 cows were randomly selected from a 940-cow spring calving dairy farm in the North Island of New Zealand; 18 were allocated to the ultrasound hoof-trimming group and 20 were allocated to the ultrasound non-trimming group. Starting in May 2018, at the end of the 2017/18 lactation, ultrasound measurements of DDP of the right hind hoof were made on all 38 cows, and the hindlimbs of the trimming group cows were trimmed by an experienced professional hoof trimmer using the five-step Dutch method. This was repeated in October 2018 (early lactation) and May 2019 (late lactation). After calving, the cows were locomotion scored fortnightly until the end of lactation using the 4-point (0-3) scale DairyNZ system. The effect of DDP on the interval between calving and the first locomotion scores ≥1 and ≥2 was assessed using Cox proportional hazards models, and the association between trimming and DDP was explored using linear mixed models. The results suggest that DDP has no effect on the time to locomotion scores ≥1 or ≥2, although the wide confidence intervals of the latter suggest that more data are needed before any definitive conclusions can be drawn. The study failed to find any clinically important impact of prophylactic trimming on DDP. This is likely related to the finding that cows with the highest DDP at the first trimming were identified by the hoof trimmer as those needing the most trimming. The results of this study thus suggest that if the Dutch five-step method is properly applied, it is unlikely to affect sole thickness over the short-to-medium term in pasture-based cattle., Competing Interests: The authors declare no conflicts of interest.

Earlier detection followed by efficient treatment can reduce the impact of lameness. Currently, locomotion scoring (LS) is the most widely used method of early detection but has significant limitations in pasture-based cattle and is not commonly used routinely in New Zealand. Scoring in the milking parlour may be more achievable, so this study compared an in-parlour scoring (IPS) technique with LS in pasture-based dairy cows. For nine months on two dairy farms, whole herd LS (4-point 0−3 scale) was followed 24 h later by IPS, with cows being milked. Observed for shifting weight, abnormal weight distribution, swollen heel or hock joint, and overgrown hoof. Every third cow was scored. Sensitivity and specificity of individual IPS indicators and one or more, two or more or three positive indicators for detecting cows with locomotion scores ≥ 2 were calculated. Using a threshold of two or more positive indicators were optimal (sensitivity > 92% and specificity > 98%). Utilising the IPS indicators, a decision tree machine learning procedure classified cows with locomotion score class ≥2 with a true positive rate of 75% and a false positive rate of 0.2%. IPS has the potential to be an alternative to LS on pasture-based dairy farms.

Competing Interests: W. Fraser SymmansStock and Other Ownership Interests: ISIS Pharmaceuticals, Delphi Diagnostics, Eiger BioPharmaceuticalsConsulting or Advisory Role: MerckResearch Funding: Pfizer (Inst)Patents, Royalties, Other Intellectual Property: Intellectual property, Intellectual Property (expired)Uncompensated Relationships: Delphi DiagnosticsOpen Payments Link: https://openpaymentsdata.cms.gov/physician/256534 Charles M. PerouLeadership: GeneCentricStock and Other Ownership Interests: BioClassifier, GeneCentric, Reveal GenomicsConsulting or Advisory Role: BioClassifier, GeneCentric, NanoString Technologies, Veracyte, Reveal GenomicsPatents, Royalties, Other Intellectual Property: Royalties from PAM50 breast cancer gene patent application and from the lung gene signature patent James S. BlachlyConsulting or Advisory Role: AstraZeneca, AbbVie, Kite, a Gilead company, AstraZeneca, Astellas Pharma, Innate PharmaPatents, Royalties, Other Intellectual Property: Sequencing Technology patent pending (Inst)Travel, Accommodations, Expenses: Oxford Nanopore Technologies James ChenEmployment: TempusConsulting or Advisory Role: Syapse, TempusSpeakers' Bureau: Foundation MedicineResearch Funding: EisaiPatents, Royalties, Other Intellectual Property: MatchTX Benjamin G. VincentStock and Other Ownership Interests: GeneCentricConsulting or Advisory Role: GeneCentricResearch Funding: Merck (Inst) Daniel G. StoverThis author is a member of the JCO Clinical Cancer Informatics Editorial Board. Journal policy recused the author from having any role in the peer review of this manuscript.Consulting or Advisory Role: NovartisNo other potential conflicts of interest were reported.

During early lactation, most dairy cows experience negative energy balance (NEB). Failure to cope with this NEB, however, can place cows at greater risk of developing metabolic disease. Our objective was to characterise, retrospectively, lying behaviour and activity of grazing dairy cows grouped according to blood non-esterified fatty acids (NEFAs) and β-hydroxybutyrate (BHB) as indicators of postpartum metabolic state. Blood was sampled weekly for up to 4 weeks precalving, on the day of calving (day 0), daily between 1 and 4 days postcalving, and then at least weekly between week 1 and week 5 postcalving for analysis of plasma NEFAs and BHB concentrations. Two hundred and forty-four multiparous Holstein-Friesian and Holstein-Friesian × Jersey cows were classified into one of three metabolic status groups based on maximum blood NEFAs and BHB concentrations during week 1 and 2 postcalving. A cow was classified as having either: (1) low NEFAs and low BHB (Lo-Lo; n = 78), when all blood samples were <1.0 mmol/L for NEFAs and ≤1.0 mmol/L for BHB during the first 2 weeks postcalving; (2) high NEFAs and low BHB (Hi-Lo; n = 134), when blood NEFAs were ≥1.0 mmol/L and blood BHB was ≤1.0 mmol/L at the same sampling time point during the first 2 weeks postcalving; or (3) high NEFAs and high BHB (Hi-Hi; n = 32), when blood NEFAs were ≥1.0 mmol/L and blood BHB was ≥1.2 mmol/L at the same sampling time point during the first 2 weeks postcalving. Accelerometers (IceTag or IceQube devices; IceRobotics Ltd.) were used to monitor lying and activity behaviours peripartum (-21 to +35 days relative to calving). Changes in lying behaviour and activity occurred before the mean day that cows were classified Hi-Hi and Hi-Lo (2.2 and 3.5 d postcalving, respectively). Up to 3 weeks preceding calving, Hi-Hi cows were more active, had fewer daily lying bouts (LBs), and spent less time lying than Lo-Lo cows. In addition, Hi-Hi cows had fewer daily LBs and were less active up to 4 weeks postcalving than Lo-Lo cows, but these differences were biologically small. Groups of grazing cows classified as experiencing a more severe metabolic challenge behave differently up to 3 weeks precalving than their herdmates with lower blood NEFAs and BHB postcalving. These altered behaviours may allow identification of individual cows at risk of a metabolic challenge, but further research is required., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

A safe, efficacious, and clinically applicable immunosuppressive regimen is necessary for islet xenotransplantation to become a viable treatment option for diabetes. We performed intraportal transplants of wild-type adult porcine islets in 25 streptozotocin-diabetic cynomolgus monkeys. Islet engraftment was good in 21, partial in 3, and poor in 1 recipient. Median xenograft survival was 25 days with rapamycin and CTLA4Ig immunosuppression. Adding basiliximab induction and maintenance tacrolimus to the base regimen significantly extended median graft survival to 147 days (p < .0001), with three animals maintaining insulin-free xenograft survival for 265, 282, and 288 days. We demonstrate that this regimen suppresses non-Gal anti-pig antibody responses, circulating effector memory T cell expansion, effector function, and infiltration of the graft. However, a chronic systemic inflammatory state manifested in the majority of recipients with long-term graft survival indicated by increased neutrophil to lymphocyte ratio, IL-6, MCP-1, CD40, and CRP expression. This suggests that this immunosuppression regimen fails to regulate innate immunity and resulting inflammation is significantly associated with increased incidence and severity of adverse events making this regimen unacceptable for translation. Additional studies are needed to optimize a maintenance regimen for regulating the innate inflammatory response., (© 2021 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Key Points: Two groups of inexperienced brain-computer interface users underwent a purely mental EEG-BCI session that rapidly impacted on their brain. Modulations in structural and functional MRI were found after only 1 h of BCI training. Two different types of BCI (based on motor imagery or visually evoked potentials) were employed and analyses showed that the brain plastic changes are spatially specific for the respective neurofeedback. This spatial specificity promises tailored therapeutic interventions (e.g. for stroke patients)., Abstract: A brain-computer-interface (BCI) allows humans to control computational devices using only neural signals. However, it is still an open question, whether performing BCI also impacts on the brain itself, i.e. whether brain plasticity is induced. Here, we show rapid and spatially specific signs of brain plasticity measured with functional and structural MRI after only 1 h of purely mental BCI training in BCI-naive subjects. We employed two BCI approaches with neurofeedback based on (i) modulations of EEG rhythms by motor imagery (MI-BCI) or (ii) event-related potentials elicited by visually targeting flashing letters (ERP-BCI). Before and after the BCI session we performed structural and functional MRI. For both BCI approaches we found increased T1-weighted MR signal in the grey matter of the respective target brain regions, such as occipital/parietal areas after ERP-BCI and precuneus and sensorimotor regions after MI-BCI. The latter also showed increased functional connectivity and higher task-evoked BOLD activity in the same areas. Our results demonstrate for the first time that BCI by means of targeted neurofeedback rapidly impacts on MRI measures of brain structure and function. The spatial specificity of BCI-induced brain plasticity promises therapeutic interventions tailored to individual functional deficits, for example in patients after stroke., (© 2019 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Objective: According to the European Reference Organization for Quality Assured Breast Cancer Screening and Diagnostic Services (EUREF) image quality in mammography is assessed by recording and analyzing a set of images of the CDMAM phantom. The EUREF procedure applies an automated analysis combining image registration, signal detection and nonlinear fitting. We present a proof of concept for an end-to-end deep learning framework that assesses image quality on the basis of single images as an alternative., Methods: Virtual mammography is used to generate a database with known ground truth for training a regression convolutional neural net (CNN). Training is carried out by continuously extending the training data and applying transfer learning., Results: The trained net is shown to correctly predict the image quality of simulated and real images. Specifically, image quality predictions on the basis of single images are of similar quality as those obtained by applying the EUREF procedure with 16 images. Our results suggest that the trained CNN generalizes well., Conclusion: Mammography image quality assessment can benefit from the proposed deep learning approach., Significance: Deep learning avoids cumbersome pre-processing and allows mammography image quality to be estimated reliably using single images.

Until recently, animal behavior has been studied through close and extensive observation of individual animals and has relied on subjective assessments. Wearable technologies that allow the automation of dairy cow behavior recording currently dominate the precision dairy technology market. Wearable accelerometers provide new opportunities in animal ethology using quantitative measures of dairy cow behavior. Recent research developments indicate that quantitative measures of behavior may provide new objective on-farm measures to assist producers in predicting, diagnosing, and managing disease or injury on farms and allowing producers to monitor cow comfort and estrus behavior. These recent research developments and a large increase in the availability of wearable accelerometers have led to growing interest of both researchers and producers in this technology. This review aimed to summarize the studies that have validated lying behavior derived from accelerometers and to describe the factors that should be considered when using leg-attached accelerometers and neck-worn collars to describe lying behavior (e.g., lying time and lying bouts) in dairy cows for research purposes. Specifically, we describe accelerometer technology, including the instrument properties and methods for recording motion; the raw data output from accelerometers; and methods developed for the transformation of raw data into meaningful and interpretable information. We highlight differences in validation study outcomes for researchers to consider when developing their own experimental methodology for the use of accelerometers to record lying behaviors in dairy cows. Finally, we discuss several factors that may influence the data recorded by accelerometers and highlight gaps in the literature. We conclude that researchers using accelerometers to record lying behaviors in dairy cattle should (1) select an accelerometer device that, based on device attachment and sampling rate, is appropriate to record the behavior of interest; (2) account for cow-, farm-, and management-related factors that could affect the lying behaviors recorded; (3) determine the appropriate editing criteria for the accurate interpretation of their data; (4) support their chosen method of recording, editing, and interpreting the data by referencing an appropriately designed and accurate validation study published in the literature; and (5) report, in detail, their methodology to ensure others can decipher how the data were captured and understand potential limitations of their methodology. We recommend that standardized protocols be developed for collecting, analyzing, and reporting lying behavior data recorded using wearable accelerometers for dairy cattle., (© 2020, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Hypocalcemia is a common metabolic disorder of transition dairy cows that is considered a gateway disease, increasing the risk of other health disorders and reducing cow performance. Clinical milk fever is associated with long periods of recumbency, and it is plausible that cows experiencing non-paretic hypocalcemia may spend more time lying; hence, lying behavior and activity measures may be useful in identifying at-risk cows. The objective of this study was to describe associations among blood calcium (Ca) status at calving and lying behavior and activity measures during the transition period in grazing dairy cows. Blood was sampled on the day of calving (d 0), and d 1, 2, 3, and 4 postcalving, and analyzed for total plasma Ca concentration. Twenty-four multiparous Holstein-Friesian and Holstein-Friesian × Jersey grazing dairy cows were classified, retrospectively, as clinically hypocalcemic (CLIN; blood Ca ≤ 1.4 mmol/L at 1 or more consecutive samplings within 48 h postcalving, but without parturient paresis). These cows were pair-matched (using milk production potential from their estimated breeding value for milk protein, mean body weight at wk -5 and -6 precalving, and, where possible, parity) with 24 cows classified as subclinically hypocalcemic (SUB; blood Ca > 1.4 and < 2.0 mmol/L at 2 consecutive samplings within 48 h postcalving), and 24 cows classified as normocalcemic (NORM; blood Ca ≥ 2.0 mmol/L at 3 consecutive samplings within 72 h postcalving). Lying behavior and activity were monitored using triaxial accelerometers from -21 to +35 d relative to calving. Data were summarized to calculate daily lying time (h/d), daily number of lying bouts (LB; no./d), mean LB duration (min/bout), and the number of steps taken (steps/d). On d 0, the CLIN group were less active and spent approximately 2.6 h longer lying than the SUB and NORM groups, particularly between 0200 and 1400 h. On d 0, the NORM group had fewer LB (16.3/d) than the SUB and CLIN groups (18.2 and 19.2/d, respectively). These differences in behavior were no longer detected 2 d postcalving, and no further differences were observed. The day before calving, the CLIN group spent 1.4 h longer lying down than did the SUB and NORM groups. Further, the relative change in steps from a precalving baseline period (d -14 to -7) until d 0 was positively, linearly associated with blood Ca concentration within 24 h postcalving. Future work should consider daily and temporal changes in behavior in individual cows to determine the potential for these measures to allow early detection of hypocalcemia., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Background: Xenotransplantation of porcine islets has emerged in recent decades as a potential treatment for type 1 diabetes (T1D). Current methods of detection, indicative of successful engraftment, occur downstream of actual islet death. Epigenetic biomarkers can be detected in circulating cell-free DNA (cfDNA) to provide an earlier indication of graft dysfunction., Aims: The present study identified a biomarker of islet death using differential methylation of the insulin gene, INS, originating from β-cells in porcine islets., Materials & Methods: Pyrosequencing primers specific for porcine INS were designed to quantify hypomethylation along 12 cysteine-guanine dinucleotide (CpG) sites, including three sites in the cyclic adenosine monophosphate (cAMP) response element (CRE) binding protein 2 (CRE2) binding region of the 5' untranslated region (UTR) and nine sites within intron 2., Results: PCR amplification of bisulfite-converted DNA combined with pyrosequencing data support the conclusion that hypomethylated porcine INS is specific to islet origin., Conclusion: Moreover, the results of this study indicate a highly specific epigenetic biomarker, capable of detecting a single islet, supporting the measurement of cfDNA as a biomarker for transplanted islet death. Defining the epigenetic characteristics of porcine-derived islets within cfDNA will be crucial to develop a better understanding of graft survival immunology for transplantation., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The aim of this study was to investigate the efficacy of dietary endotoxin binders [bentonite (BEN) and Saccharomyces cerevisiae cell wall (SCW)] on acute-phase protein (APP) response and liver function in cows during the transition period. Twenty-four multiparous Holstein cows were randomly assigned to one of four treatment groups. The experimental groups consisted of (1) the basal diet (BD) + SCW, (2) BD + SCW + BEN, (3) BD + BEN, and (4) BD (control). Blood samples were taken at 1, 3 and 4 weeks before and 1 and 3 weeks after parturition and serum concentrations of non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHBA), glucose, haptoglobin (Hp), serum amyloid A(SAA), albumin, g-glutamyl transferase (GGT), aspartate aminotransferase (AST), cholesterol, iron, and lipopolysaccharide (LPS) were measured. The concentrations of LPS, SAA, albumin, and Hp in the blood were within reference range at all times. The level of blood LPS was not high enough to initiate an APP response. Mean BHBA concentration was highest at 1 week after calving. For NEFA, the pattern was similar, with a peak at 1 week after calving. Cholesterol concentration was lower in the SCW group, probably due to a lower lipoprotein concentration. Mean AST concentration was highest at 1 week after calving, especially in the SCW + BEN group. The results of a current study showed that, if the carbohydrate level is not high in the diet to cause rumen acidosis, it is not profitable to supplement BEN and SCW for adsorbing endotoxins in the diet, in transition cows.

Background: There is currently a shortage of human donor pancreata which limits the broad application of islet transplantation as a treatment for type 1 diabetes. Porcine islets have demonstrated potential as an alternative source, but a study evaluating islets from different donor ages under unified protocols has yet to be conducted., Methods: Neonatal porcine islets (NPI; 1-3 days), juvenile porcine islets (JPI; 18-21 days), and adult porcine islets (API; 2+ years) were compared in vitro, including assessments of oxygen consumption rate, membrane integrity determined by FDA/PI staining, β-cell proliferation, dynamic glucose-stimulated insulin secretion, and RNA sequencing., Results: Oxygen consumption rate normalized to DNA was not significantly different between ages. Membrane integrity was age dependent, and API had the highest percentage of intact cells. API also had the highest glucose-stimulated insulin secretion response during a dynamic insulin secretion assay and had 50-fold higher total insulin content compared to NPI and JPI. NPI and JPI had similar glucose responsiveness, β-cell percentage, and β-cell proliferation rate. Transcriptome analysis was consistent with physiological assessments. API transcriptomes were enriched for cellular metabolic and insulin secretory pathways, while NPI exhibited higher expression of genes associated with proliferation., Conclusions: The oxygen demand, membrane integrity, β-cell function and proliferation, and transcriptomes of islets from API, JPI, and NPI provide a comprehensive physiological comparison for future studies. These assessments will inform the optimal application of each age of porcine islet to expand the availability of islet transplantation., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Islet transplantation (ITx) is an emerging and promising therapy for patients with uncontrolled type 1 diabetes. The islet isolation and purification processes require exposure to extended cold ischemia, warm-enzymatic digestion, mechanical agitation, and use of damaging chemicals for density gradient separation (DG), all of which reduce viable islet yield. In this paper, we describe initial proof-of-concept studies exploring quadrupole magnetic separation (QMS) of islets as an alternative to DG to reduce exposure to these harsh conditions. Three porcine pancreata were split into two parts, the splenic lobe (SPL) and the combined connecting/duodenal lobes (CDL), for paired digestions and purifications. Islets in the SPL were preferentially labeled using magnetic microparticles (MMPs) that lodge within the islet microvasculature when infused into the pancreas and were continuously separated from the exocrine tissue by QMS during the collection phase of the digestion process. Unlabeled islets from the CDL were purified by conventional DG. Islets purified by QMS exhibited significantly improved viability (measured by oxygen consumption rate per DNA, p < 0.03) and better morphology relative to control islets. Islet purification by QMS can reduce the detrimental effects of prolonged exposure to toxic enzymes and density gradient solutions and substantially improve islet viability after isolation.

Background: Reliable in vitro islet quality assessment assays that can be performed routinely, prospectively, and are able to predict clinical transplant outcomes are needed. In this paper we present data on the utility of an assay based on cellular oxygen consumption rate (OCR) in predicting clinical islet autotransplant (IAT) insulin independence (II). IAT is an attractive model for evaluating characterization assays regarding their utility in predicting II due to an absence of confounding factors such as immune rejection and immunosuppressant toxicity., Methods: Membrane integrity staining (FDA/PI), OCR normalized to DNA (OCR/DNA), islet equivalent (IE) and OCR (viable IE) normalized to recipient body weight (IE dose and OCR dose), and OCR/DNA normalized to islet size index (ISI) were used to characterize autoislet preparations (n = 35). Correlation between pre-IAT islet product characteristics and II was determined using receiver operating characteristic analysis., Results: Preparations that resulted in II had significantly higher OCR dose and IE dose (p<0.001). These islet characterization methods were highly correlated with II at 6-12 months post-IAT (area-under-the-curve (AUC) = 0.94 for IE dose and 0.96 for OCR dose). FDA/PI (AUC = 0.49) and OCR/DNA (AUC = 0.58) did not correlate with II. OCR/DNA/ISI may have some utility in predicting outcome (AUC = 0.72)., Conclusions: Commonly used assays to determine whether a clinical islet preparation is of high quality prior to transplantation are greatly lacking in sensitivity and specificity. While IE dose is highly predictive, it does not take into account islet cell quality. OCR dose, which takes into consideration both islet cell quality and quantity, may enable a more accurate and prospective evaluation of clinical islet preparations.

Background: The amount and condition of exocrine impurities may affect the quality of islet preparations, especially during culture. In this study, the objective was to determine the oxygen demand and viability of islet and acinar tissue post-isolation and whether they change disproportionately while in culture., Method: We compared the oxygen consumption rate (OCR) normalized to DNA (OCR/DNA, a measure of fractional viability in units of nmol/min/mg DNA), and the percent change in OCR and DNA recoveries between adult porcine islet and acinar tissue from the same preparation (paired) over 6-9 days of standard culture. Paired comparisons were done to quantify differences in OCR/DNA between islet and acinar tissue from the same preparation, at specified time points during culture., Results: The mean (±SE) OCR/DNA was 74.0 ± 11.7 units higher for acinar (vs islet) tissue on the day of isolation (n = 16, P < .0001), but 25.7 ± 9.4 units lower after 1 day (n = 8, P = .03), 56.6 ± 11.5 units lower after 2 days (n = 12, P = .0004), and 65.9 ± 28.7 units lower after 8 days (n = 4, P = .2) in culture. DNA and OCR recoveries decreased at different rates for acinar versus islet tissue over 6-9 days in culture (n = 6). DNA recovery decreased to 24 ± 7% for acinar and 75 ± 8% for islets (P = .002). Similarly, OCR recovery decreased to 16 ± 3% for acinar and remained virtually constant for islets (P = .005)., Conclusion: Differences in the metabolic profile of acinar and islet tissue should be considered when culturing impure islet preparations. OCR-based measurements may help optimize pre-islet transplantation culture protocols., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The scarcity of human cadaveric pancreata limits large-scale application of islet transplantation for patients with diabetes. Islets isolated from pathogen-free pigs provide an economical and abundant alternative source assuming immunologic barriers are appropriate. Membrane receptors involved in insulin secretion that also have potential as imaging targets were investigated in isolated porcine islets. Quantitative (q)PCR revealed that porcine islets express mRNA transcripts for sulfonylurea receptor 1 (Sur1), inward rectifying potassium channel (Kir6.2, associated with Sur1), glucagon-like peptide 1 receptor (GLP1R), and adrenergic receptor alpha 2A (ADRα2A). Receptor function was assessed in static incubations with stimulatory glucose concentrations, and in the presence of receptor agonists. Glibenclamide, an anti-diabetic sulfonylurea, and exendin-4, a GLP-1 mimetic, potentiated glucose-stimulated insulin secretion >2-fold. Conversely, epinephrine maximally reduced insulin secretion 72 ± 9% (P < 0.05) and had a half maximal inhibitory concentration of 60 nm in porcine islets (95% confidence interval of 45-830 nm). The epinephrine action was inhibited by the ADRα2A antagonist yohimbine. Our findings demonstrate that porcine islets express and are responsive to both stimulatory and inhibitory membrane localized receptors, which can be used as imaging targets after transplantation or to modify insulin secretion., (© 2014 John Wiley & Sons A/S Published by John Wiley & Sons Ltd.)

Background: Replacement of β-cells with the use of isolated islet allotransplantation (IT) is an emerging therapy for type 1 diabetics with hypoglycemia unawareness. The current standard protocol calls for a 36-72-hour culture period before IT. We examined 13 clinical islet preparations with ≥2 purity fractions to determine the effect of culture on viability., Methods: After standard islet isolation and purification, pure islet fractions were placed at 37°C with 5% CO2 for 12-24 hours and subsequently moved to 22°C, whereas less pure fractions were cultured at 22°C for the entire duration. Culture density was targeted at a range of 100-200 islet equivalents (IEQ)/cm(2) adjusted for purity. Islets were assessed for purity (dithizone staining), quantity (pellet volume and DNA), and viability (oxygen consumption rate normalized to DNA content [OCR/DNA] and membrane integrity)., Results: Results indicated that purity was overestimated, especially in less pure fractions. This was evidenced by significantly larger observed pellet sizes than expected and tissue amount as quantified with the use of a dsDNA assay when available. Less pure fractions showed significantly lower OCR/DNA and membrane integrity compared with pure. The difference in viability between the 2 purity fractions may be due to a variety of reasons, including hypoxia, nutrient deficiency, toxic metabolite accumulation, and/or proteolytic enzymes released by acinar tissue impurities that are not neutralized by human serum albumin in the culture media., Conclusions: Current clinical islet culture protocols should be examined further, especially for less pure fractions, to ensure the maintenance of viability before transplantation. Even though relatively small, the difference in viability is important because the amount of dead or dying tissue introduced into recipients may be dramatically increased, especially with less pure preparations., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Stress conditions generated throughout pancreatic islet processing initiate the activation of pro-inflammatory pathways and beta-cell destruction. Our goal is to identify relevant and preferably beta-specific markers to assess the activation of beta-cell stress and apoptotic mechanisms, and therefore the general quality of the islet preparation prior to transplantation. Protein expression and activation were analyzed by Western blotting and kinase assays. ATP measurements were performed by a luminescence-based assay. Oxygen consumption rate (OCR) was measured based on standard protocols using fiber optic sensors. Total RNA was used for gene expression analyzes. Our results indicate that pancreas digestion initiates a potent stress response in the islets by activating two stress kinases, c-Jun N-terminal Kinase (JNK) and p38. JNK1 protein levels remained unchanged between different islet preparations and following culture. In contrast, levels of JNK3 increased after islet culture, but varied markedly, with a subset of preparations bearing low JNK3 expression. The observed changes in JNK3 protein content strongly correlated with OCR measurements as determined by the Spearman's rank correlation coefficient rho [Formula: see text] in the matching islet samples, while inversely correlating with c-fos mRNA expression [Formula: see text]. In conclusion, pancreas digestion recruits JNK and p38 kinases that are known to participate to beta-cell apoptosis. Concomitantly, the islet isolation alters JNK3 and c-fos expression, both strongly correlating with OCR. Thus, a comparative analysis of JNK3 and c-fos expression before and after culture may provide for novel markers to assess islet quality prior to transplantation. JNK3 has the advantage over all other proposed markers to be islet-specific, and thus to provide for a marker independent of non-beta cell contamination.

Background: The manufacturing process of islets includes a culture step which was originally introduced to ease the logistics of procedures in preparing the graft and transplant recipient. It has been suggested that culture at room temperature has an advantage over culture at 37 °C, in part by reducing immunogenicity via preferential elimination of contaminating cells (such as passenger leukocytes) within islets. We investigated this using islets isolated from pancreata of adult pigs., Methods: Porcine islets were isolated from three donors and cultured at 37 °C for 1 day, and then under three different conditions: 37 °C for 6 days (condition A); 22 °C for 6 days (condition B); or 22 °C for 5 days followed by 37 °C for 1 day (condition C). Recovery was assessed by DNA measurement, viability by oxygen consumption rate normalized for DNA (OCR/DNA), and gene expression by RT-PCR for a series of 9 lymphocyte markers, 11 lymphokines and chemokines, and 14 apoptotic and stress markers., Results: Post-culture islet recoveries were similar for the three culture conditions. Average OCR/DNA values were 129-159 nmol/min·mgDNA before culture, and 259-291, 204-212, and 207-228 nmol/min·mgDNA, respectively, for culture under conditions A, B, and C, respectively. Irrespective of culture condition, examined gene expression in all three series of lymphocyte markers, lymphokines and chemokines, and apoptotic and stress markers manifested a statistically significant decrease upon culture for 7 days. This decrease was most dramatic for condition A: in particular, most of lymphocyte markers showed a >10-fold reduction and also six markers in the lymphokine and chemokine series; these reductions are consistent with the elimination of immune cells present within islets during culture. The reduction was less for apoptotic and stress markers. For culture under condition B, the reduction in gene expression was less, and culture under condition C resulted in gene expression levels similar to those under condition A: this indicates that 24 h at 37 °C is sufficient to re-equilibrate gene expression levels from those in islets cultured at 22 °C to those in islets cultured at 37 °C. Results were consistent among the preparations from the three donors., Conclusions: Culture of porcine islets at 37 °C provides benefits over culture at 22 °C with respect to OCR/DNA outcomes and reduced expression of genes encoding lymphocyte markers, lymphokines and chemokines, and markers for apoptosis and stress., (© 2013 John Wiley & Sons A/S.)

Porcine islet xenotransplantation is considered a potential cell-based therapy for type 1 diabetes. It is currently being evaluated in diabetic nonhuman primates (NHP) to assess safety and efficacy of the islet product. However, due to a variety of distinct differences between the respective species, including the insulin secretory characteristics of islets, the suitability and predictive value of the preclinical model in the extrapolation to the clinical setting remain a critical issue. Islets isolated from human (n = 3), NHP (n = 2), adult pig (AP, n = 3), and juvenile pig (JP, n = 4) pancreata were perifused with medium at basal glucose (2.5 mm) followed by high glucose (16.7 mm) concentrations. The total glucose-stimulated insulin secretion (GSIS) was calculated from generated insulin secretion profiles. Nonhuman primate islets exhibited GSIS 3-fold higher than AP islets, while AP and JP islets exhibited GSIS 1/3 and 1/30 of human islets, respectively. The insulin content of NHP and AP islets was similar to that of human islets, whereas that of JP islets was 1/5 of human islets. Despite the fact that human, NHP, and AP islets contain similar amounts of insulin, the much higher GSIS for NHP islets than for AP and JP islets suggests the need for increased dosing of islets from JP and AP in pig-to-NHP transplantation. Porcine islet xenotransplantation to humans may require significantly higher dosing given the lower GSIS of AP islets compared to human islets., (© 2013 John Wiley & Sons A/S.)

Background: Porcine islet transplantation is emerging as an attractive option for the treatment of patients with type 1 diabetes, with the possibility of providing islets of higher and more consistent quality and in larger volumes than available from human pancreata. The use of encapsulated neonatal porcine islets (ENPI) is appealing because it can address islet supply limitations while reducing the need for anti-rejection therapy. Pre-transplant characterization of ENPI viability and potency is an essential component of the production process. We applied the validated assay for oxygen consumption rate normalized for DNA content (OCR/DNA) to characterize ENPI viability., Methods: ENPI of low viscosity and high m alginate were prepared according to standard methods and characterized at various culture time points up to 5 weeks., Results: The OCR/DNA (nmol/min·mgDNA ± SEM) of ENPI (235 ± 10, n = 9) was comparable to that of free NPI (255 ± 14, n = 13). After encapsulation, NPI OCR/DNA was sustained over a culture period of up to 5 weeks. The average OCR/DNA of ENPI cultured longer than 9 days was higher than that of freshly encapsulated NPI., Conclusion: This is the first characterization of ENPI by a validated and more sensitive method for product viability. The NPI encapsulation process does not compromise viability as measured by OCR/DNA, and ENPI can be cultured for up to 5 weeks with maintenance of viability. ENPI meet or exceed current adult porcine islet product release criteria (established at the University of Minnesota) for preclinical xenotransplantation in terms of OCR/DNA., (© 2012 John Wiley & Sons A/S.)

Culture of human islets before clinical transplantation or distribution for research purposes is standard practice. At the time the Edmonton protocol was introduced, clinical islet manufacturing did not include culture, and human serum albumin (HSA), instead of fetal bovine serum (FBS), was used during other steps of the process to avoid the introduction of xenogeneic material. When culture was subsequently introduced, HSA was also used for medium supplementation instead of FBS, which was typically used for research islet culture. The use of HSA as culture supplement was not evaluated before this implementation. We performed a retrospective analysis of 103 high-purity islet preparations (76 research preparations, all with FBS culture supplementation, and 27 clinical preparations, all with HSA supplementation) for oxygen consumption rate per DNA content (OCR/DNA; a measure of viability) and diabetes reversal rate in diabetic nude mice (a measure of potency). After 2-day culture, research preparations exhibited an average OCR/DNA 51% higher (p < 0.001) and an average diabetes reversal rate 54% higher (p < 0.05) than clinical preparations, despite 87% of the research islet preparations having been derived from research-grade pancreata that are considered of lower quality. In a prospective paired study on islets from eight research preparations, OCR/DNA was, on average, 27% higher with FBS supplementation than that with HSA supplementation (p < 0.05). We conclude that the quality of clinical islet preparations can be improved when culture is performed in media supplemented with serum instead of albumin.

The demand for high-quality islets for transplantation in type I diabetics will increase as the current clinical trials transition into standard of care. The mode of preservation of donor pancreata is critical to this mission since islets are very sensitive to ischemic injury. Hypothermic perfusion preservation (HPP) is being investigated for extended pancreas preservation in light of the beneficial effects reported for other organs. The present pilot study aimed to establish the potency of porcine islets isolated from pancreata after 24 h of HPP at 4-8°C. The study design included a split-lobe pancreas model that permitted paired comparisons of islets isolated from 24-h HPP splenic lobes with nonperfused, fresh control duodenal/connecting lobes stored at 4°C for <3 h. Prior to transplantation, islet viability was assessed in vitro using the ratio of oxygen consumption rate to DNA (OCR/DNA) assay and correlated with subsequent in vivo function by transplantation in diabetic immunodeficient mice. The OCR/DNA (mean ± SD) measured after 7 days of culture and immediately prior to transplantation for islets from the 24-h HPP group was 269 ± 19 nmol/min/mg DNA, which was higher but not statistically different to the mean of 236 ± 43 for the counterpart control group. All four nude mice transplanted with islets from the 24-h HPP group showed diabetes reversal, compared with five of six transplants from the control group. In conclusion, islets isolated from adult porcine pancreata after 24-h HPP exhibited high viability as measured by OCR/DNA and were able to consistently reverse diabetes in a nude mouse bioassay.

The mechanisms by which HIV-1 affects thymopoiesis were determined by preincubating CD34+ cells or cultured thymic epithelial (CTE) cells with lymphotropic (T-) and monotropic (M-) strains of HIV-1 in an in vitro CTE organ and CD34+ cell coculture model that allows for analysis of development of thymocytes and mature T cells. When purified CD34+ cells were precultured with either T- or M-tropic strains of HIV-1, thymopoiesis was impaired in a two-week coculture manifested by decreased cell number of thymocytes generated. However, the percentages of thymocyte subpopulations were comparable to control uninfected cocultures. Furthermore, HIV infection of thymocytes was predominantly observed in the CD44+CD3- population. However, in a four-week coculture experiment, HIV infection and depletion of more mature thymocytes were also observed. When CTE cells were preincubated with T- and M-tropic strains of HIV before addition of CD34+ cells, the number of thymocytes and subpopulations of thymocytes at early and later stages of maturation were markedly decreased. Furthermore, CD34+ and CD44+CD3- cells become HIV-infected. In summary, HIV-1 infection inhibited thymocyte maturation at early stages of thymocyte maturation CD44+CD25-CD3-. In addition, HIV also depleted later stages of CD4+ thymocyte subpopulations.

The effect of thymosin-alpha1 on thymopoiesis is largely unknown. Thymosin is found in the cortical and medullary thymic epithelia, as well as in nurse cells; thus, it is hypothesized that thymosin may affect both early and late stage of thymocyte maturation. In this study, the effect of thymosin-alpha1 on thymopoiesis was determined by coculturing in vitro CD34+ stem cells (SC) with allogeneic cultured thymic epithelia fragments (CTEF) for 1-4 weeks and analyzing T-cell maturation by flow cytometry. Thymosin-alpha1 significantly enhanced the cell number (e.g., proliferation) of mononuclear cells obtained at 2 and 4 weeks of the SC-CTEF cocultures (P < 0.01 and < 0.05, respectively). In particular, thymosin-alpha1 stimulated expression of CD3+ cells at 3 and 4 weeks (P < 0.05). The predominant subpopulation increased by thymosin stimulation was single positive mature CD4+ cells, which was confirmed to occur within the SC-CTEF thymic organ tissue by laser confocal immunofluorescence microscopy. Thymosin stimulation tended to enhance IL-7 synthesis, critical cytokine in the maturation of thymocytes. In summary, this is the first study to demonstrate that thymosin-alpha1 enhanced thymopoiesis of CD34+ stem cells in humans using an in vitro model of differentiation using stem cells and cultured thymic epithelial fragments cocultures. Furthermore, the thymosin significantly increased expression of CD3+4+ T cells.

Objective: To characterize distinctive lymphoid cell populations in the synovial fluid (SF) of patients with juvenile rheumatoid arthritis (JRA) that have the specific ability to display monocytic markers when cultured in vitro., Methods: Mononuclear cells obtained from SF of patients with JRA and depleted of adherent macrophages were cultured in vitro in RPMI 1640 medium supplemented with only fetal calf serum (FCS). Phenotypic evaluation of these cells was by flow cytometry and immunohistochemical analysis was by specific fluorochrome labeled antibodies., Results: T cells from a JRA subgroup displayed some typical macrophage attributes, i.e., abundant cytoplasm, adherence to plastic, and phagocytosis of latex beads when cultured in vitro. These cells have the ability to survive in culture for several weeks in RPMI 1640 medium containing only 10% FCS. The macrophage-like T cells rosetted with sheep red blood cells and proliferated when stimulated with phytohemagglutinin or anti-CD3, indicating functional T cell responses., Conclusion: Our data indicate that a population of T cells obtained from the SF of a subgroup of patients with JRA exhibited characteristics of macrophages, yet retained their CD3 and T cell receptor expression. Whether this promiscuous behavior is caused by malignant transformation of lymphoid precursor cells or is induced by the concerted effect of a myriad of cytokines and growth factors present in the SF remains unknown. The presence of these cells in the SF of 2 patients with JRA with different onset types raises the question of their function and significance in an autoimmune disorder such as JRA.

An in vitro model of CD34+CD38- stem cell (SC) differentiation in postnatal cultured thymic epithelia fragment (CTEF) cocultures is described. Sequential phenotypic analysis of the progeny of the SC-CTEF demonstrated predominantly thymocytes and minor populations of promyelocytes, monocytes and natural killer cells. Triple-positive CD3+CD4+CD8+, double-positive CD4+CD8+, and mature single-positive CD4+ and CD8+ T cells, which were TCR alpha beta+, were identified indicating normal thymocyte maturation. In kinetic studies, mature single-positive CD4+ T cells increased from 29% of total cells at one week to 54% at four weeks of coculture. These findings demonstrate that coculture of bone marrow-derived SC and allogeneic cultured thymic epithelia in vitro results in continuous normal predominantly thymocyte differentiation. The SC-CTEF cocultures were then infected with two different strains of human immunodeficiency virus. CD4+ thymocytes were markedly decreased. However, inhibition of early thymocyte maturation steps was also suggested by the presence of increased triple-negative and CD44+CD25-CD3-thymocytes and decreased CD44+CD25+ thymocytes. This model system of thymocyte maturation will be useful in the evaluation of primary T cell immunodeficiency disorders, gene therapy of SC and pharmacological augmentation of thymic function.

An in vitro coculture model system of CD34+ stem cells and allogenic cultured thymic epithelia fragments was used to evaluate thymocyte differentiation in a 9-month-old child of Amish descent with Nezelof syndrome. Though the patient's stem cells differentiate to acquire normal expression of CD2 and CD7, later steps of maturation were abnormal. There was detectable but reduced expression of CD3 and CD4 phenotypes. CD44+ expression, however, was markedly reduced. CD44 is an adhesion molecule, interacting with the matrix ligands hyaluronan and fibronectin, and is expressed early in thymocyte differentiation and subsequently in mature T cells. It is hypothesized that abnormal expression of CD44 in a variant of severe combined immunodeficiency, Nezelof's syndrome, interferes with normal thymocyte and thymic epithelial interaction, which leads to abnormal thymocyte differentiation.

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease caused by bronchial colonization with Aspergillus fumigatus (Af) characterized by elevated serum total and Af-specific IgE levels and eosinophilia. In order to examine T-cell reactivity to Af antigens, six T-cell lines were established from the peripheral blood of patients with ABPA to Asp f I, an 18 kd protein purified from Af extracts. The Asp f I-specific T-cell lines, analyzed by flow cytometry, were 100% CD3+ CD4+. Lymphoproliferative responses of the T-cell lines were specific for Asp f I stimulation, 28,999 cpm (stimulation index = 12.2), and showed no response to tetanus toxoid stimulation, 2178 cpm (stimulation index = 1.1) (p < 0.001). Furthermore, Asp f I-stimulated lymphoproliferation was inhibited in two experiments by monoclonal anti-interleukin (IL)-4 antibody in a dose-response fashion, 78% and 84% inhibition at 5% concentration of anti-IL-4. In contrast, anti-IL-2 antibody did not inhibit Asp f I-stimulated proliferation. Asp f I-stimulated T-cell lines synthesized predominantly IL-4 (mean, 21.5 ng/ml) after 48 hours of culture, and nondetectable quantities of interferon-gamma and IL-2. In summary, Asp f I-specific T-cell lines established from patients with ABPA were characterized as being CD4+ TH2-like in their cytokine synthesis pattern, and secreted IL-4 behaved in an autocrine fashion, stimulating proliferation.

Allergic bronchopulmonary aspergillosis (ABPA) occurs with a prevalence of 5% to 15% in patients with cystic fibrosis (CF). Because of the frequent colonization with Aspergillus fumigatus (Af) in CF, the causative agent of ABPA, antibody reactivity to Af proteins is frequently observed, which obscures the diagnosis of ABPA. Patients with CF are also categorized according to the presence of positive skin test responses to Af and/or the presence of positive precipitins. In this study we used ELISA and immunoblot assay to detect IgE and IgG anti-Af antibodies in patients with CF and ABPA (n = 13) compared with other groups of patients with CF: those with positive skin test and positive precipitin results (n = 18), those with positive skin test and negative precipitin results (n = 14), those with negative skin test and positive precipitin results (n = 10), and those with negative skin test and negative precipitin results (n = 35). IgE and IgG anti-Af antibodies were significantly elevated in patients with ABPA as determined by both immunoblot assay (p < 0.01) and ELISA (p < 0.01). However, detection of Af antibodies by ELISA was more sensitive in discriminating patients with CF and ABPA from patients with CF who had positive skin test and positive precipitin results but lacked radiographic and clinical evidence of ABPA. In patients with CF and ABPA the immunoblot assays demonstrated a multitude of IgE, IgG, and IgA antibody responses to Af proteins, which ranged in molecular weight from 14 kd to greater than 106 kd. The level of IgE anti-Af antibody to individual proteins decreased during remissions of ABPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Failure of an intact ventriculoperitoneal shunt, in the absence of an overt infection, is often due to its occlusion by cellular debris and/or an abdominal pseudocyst. This failure is thought to be caused by an infection by an organism which is difficult to culture or by some poorly defined allergic response to the shunt materials. Little attention has been directed to the treatment that the shunts receive prior to implantation: specifically, their exposure to ethylene oxide as a means of sterilization. We have found ethylene oxide metabolites in the spinal fluid of children with shunt malfunction months after their systems were implanted. Many of these patients had coincident CSF eosinophilia. In addition, two of the children had detectable serum IgE antibody directed against an albumin-ethylene oxide conjugated protein. Both of these children had several shunt malfunctions within a short period, yet neither child could be shown to have a shunt infection despite multiple cultures. We therefore suggest that in some patients proteins altered by ethylene oxide incite an IgE mediated response which may lead to shunt malfunction.

Patients with cystic fibrosis frequently have pulmonary colonization with Aspergillus fumigatus (Af) and develop anti-Af immunoglobulin E (IgE) and IgG antibodies. The diagnosis of allergic bronchopulmonary aspergillosis in subjects with cystic fibrosis is difficult because of the high incidence of Af colonization, with development of humoral antibody responses. In this study, we sequentially measured serum anti-Af IgE (Af-E) and IgG (Af-G) antibodies by ELISA in subjects with cystic fibrosis. In subjects with cystic fibrosis who have allergic bronchopulmonary aspergillosis, Af-E and Af-G antibodies were significantly increased when compared with other groups of patients with cystic fibrosis who had positive skin tests or precipitins to Af (or both) (p less than 0.01, p less than 0.01, respectively). In addition, increased Af-E and Af-G levels were sometimes seen in other groups, especially subjects with cystic fibrosis who had positive Af skin tests or precipitin tests, two of whom later developed criteria diagnostic of allergic bronchopulmonary aspergillosis. Thus, serum Af-E and Af-G levels were quantitatively increased in subjects with cystic fibrosis who had allergic bronchopulmonary aspergillosis and thus adjunctive data in diagnosis. However, it also suggested that subclinical pulmonary inflammation may also occur.

Since Aspergillus fumigatus (Af)-specific and polyclonal serum IgE levels are characteristically elevated in patients with allergic bronchopulmonary aspergillosis (ABPA), we evaluated in vitro regulation of IgE synthesis in cystic fibrosis (CF) patients with ABPA. We studied 11 CF patients with ABPA, 37 patients with positive Af prick skin tests and/or IgG precipitating antibodies (ST/PPT+), and 35 patients with no humoral or skin responses to Aspergillus (ST/PPT-). Mean serum IgE concentration was significantly elevated in CF subjects with ABPA compared to ST/PPT+ and ST/PPT- patients, 2866 vs 303 and 61 IU/ml, respectively (P less than 0.01). In vitro studies demonstrated that ABPA patients' B cells spontaneously synthesized significantly increased amounts of IgE compared to ST/PPT positive and negative subjects, 1980 vs 220 and 13 pg/ml, respectively (P less than 0.01). In addition, preformed B-cell-associated IgE was also significantly elevated in ABPA subjects (P less than 0.01), indicating prior in vivo activation. Supernatant cultures of Af-stimulated T cells from ABPA subjects significantly induced allogeneic B-cell IgE synthesis compared to ST/PPT positive and negative CF subjects, 206 vs 13 and 4 pg/ml, respectively (P less than 0.01). Thus T cells stimulated with Aspergillus antigens secrete cytokines that induce B-cell IgE synthesis in ABPA subjects. B-cell IgE hyperactivity is manifested by in vivo and in vitro increased IgE concentrations. Analyses of T-cell regulation and B-cell IgE synthesis distinguish CF subjects with ABPA from Aspergillus sensitive non-ABPA subjects.

T cell cytotoxicity (CTL) to an allogeneic lymphocyte target was evaluated in patients with cystic fibrosis (CF) before and during pulmonary exacerbations (group 1) compared to another group of CF patients who had stable pulmonary disease activity during their two study periods (group 2). CTL activity was significantly decreased in group 1 subjects studied prior to their pulmonary flares and in group 2 CF patients compared to normal controls at effector:target ratios of 12.5:1 and 6.25:1 (p less than 0.05 and p less than 0.05, respectively). Furthermore, in group 1, CTL lysis was significantly decreased during pulmonary flares compared to before flares at the 25:1 and 12.5:1, effector:target, (p less than 0.05 and p less than 0.05, respectively). T suppressor cell activity as measured by effect on in vitro control B cell IgM synthesis was significantly increased during pulmonary flares (p less than 0.05). Diminished CTL may be partially responsible for persistent colonization of Pseudomonas aeruginosa in CF.

Growth curves of the 5 commonly used Ames Salmonella tester strains have been measured turbidimetrically in semi-solid agar. Lag times, doubling times and maximum cell densities have been calculated for each of the 5 strains. The time dependence of reversion has been studied in the standard plate incorporation assay using 1-h pulsed doses of (a) bromoethane, a volatile chemical mutagen, and (b) 1-h exposures to visible light. Essentially no reversion takes place during the first 4 h after plating. Reversion is detectable between hours 4 and 16. The cumulative or integrated revertants versus time curve has the characteristics of a growth curve. Conversely the derivatives of the growth curves resemble the curves obtained in the pulsed mutagenicity studies. Thus, the reversion rate in any given 1 h interval is proportional to the growth rate during that same interval. These results suggest that mutagenic chemicals must be present during the bacterial growth cycle (about 4-16 h after plating) in order to revert the tester strains. Short-lived chemical mutagens, then, should produce enhanced results if plated 6-8 h after the bacteria. We have confirmed this for N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 9-aminoacridine and 2-aminoanthracene (with S9).

Di-(2-ethylhexyl)phthalate (DEHP) produced hepatocellular carcinomas in rodents at high doses in a NTP/NCI bioassay. DEHP has not shown evidence of genotoxic activity in in vitro mutagenicity tests. We extended these studies by examining the mutagenicity of urine from rats dosed with DEHP, 2-ethylhexanol (2-EH), and several other 2-EH derived plasticizers, i.e. di-(2-ethylhexyl)adipate (DEHA), di-(2-ethylhexyl)terephthalate (DEHT) and tri-(2-ethylhexyl)trimellitate (TEHT). A modified Ames Salmonella/microsome assay was used to determine mutagenicity. Urine was pooled from male Sprague--Dawley rats dosed daily for 15 days with 2000 mg/kg of each test substance with the exception of 2-EH which was given at 1000 mg/kg. Direct plating procedures were used to determine the presence of mutagens in urine. Urine from rats dosed with 8-hydroxyquinoline was used as a positive control. There was no evidence that mutagenic substances were excreted in the urine by rats dosed with either DEHP, DEHA, DEHT, TEHT or 2-EH as determined in the presence or absence of rat liver microsomes, and with or without treatment with beta-glucuronidase/aryl sulfatase. Our findings indicate that the above test compounds were not converted to urinary metabolites that were mutagenic. These observations provide no evidence for a genotoxic mechanism for DEHP carcinogenicity in rodents.

The response of peripheral blood lymphocytes (PBL) to the antigen tetanus toxoid (TT) in patients with chronic renal failure being maintained on hemodialysis was examined. We have found that: (1) the response of patients' unfractionated PBL to TT is markedly suppressed when compared to the response of control PBL; (2) the response of patients' purified T cells and T4+ cells to TT co-cultured with 5% autologous monocytes is suppressed when compared to the response of comparable control cultures; (3) the response of patients' purified T lymphocytes co-cultured with 5% autologous monocytes is significantly enhanced over the response of patients' unfractionated PBL, and (4) the suppressed proliferation of patients' PBL to TT is not reversed by hemodialysis. Thus, the presence of suppressor monocytes and the inability of the responding T cells and accessory monocytes to react to antigen contribute to the suppressed antigen-specific T cell proliferation observed in chronic renal failure patients. These results are relevant to the suppressed cell-mediated immunity observed in uremia.

The Regional Hemophilia Center in St. Louis initiated a prospective study beginning in 1982 to measure sequentially T-cell subpopulations and in vitro lymphoproliferative responses in hemophilia A patients. In a cohort of 106 hemophiliacs, the prevalence of HIV-seropositivity increased from 46.7% in 1982 to 74.5% by 1987. There was a persistent gradual decline over time of T helper/inducer (CD4) cells in HIV-seropositive hemophiliacs (P less than .01). This was reflected by an increasing percentage of hemophiliacs with abnormally low CD4 cells (less than 2 standard deviations below the mean of normal individuals) from 6.7% in 1983 to 52.4% in 1987. Function of CD4 cells, as estimated by in vitro lymphoproliferative responses to phytohemagglutinin (PHA) and tetanus toxoid stimulations also demonstrated a decline over the same years. Lymphoproliferative responses to PHA by HIV-seropositive hemophiliacs' mononuclear cells (MNC) declined from a 90.2% normal response in 1983 to a 71.7% normal response in 1987 (P less than .05). Decreased responses to stimulation with the soluble antigen tetanus toxoid were also seen from 1983 compared with 1987 (P less than .05). This was due to an increased percentage of HIV-seropositive hemophiliacs' MNC, which were unresponsive to stimulation to tetanus toxoid (stimulation index less than 3.0) from 20.8% in 1983 to 41.0% in 1987. These findings indicate that HIV-infection was associated over time with a decline of CD4 number and function in a substantial portion of hemophiliacs.

Longitudinal immune studies of patients with hemophilia A were begun in 1982 by the Regional Hemophilia Center in St. Louis, Missouri. Serum samples collected from 74 participants between 1982 and 1985 were analyzed for antibody to human T-lymphotropic virus type III (HTLV-III)/lymphadenopathy-associated virus (LAV). The incidence of antibody to HTLV-III/LAV has increased significantly in this population of patients with hemophilia. Only one of eight hemophiliacs had detectable antibody before July 1982, whereas 88.7% (55/62) were positive in 1985. T-cell surface markers were markedly abnormal in seropositive hemophilia patients with decreased percentage and number of OKT4-positive cells compared with seronegative hemophiliacs and controls. Lymphoproliferative responses to mitogens and antigens were normal in seronegative hemophilia patients. Seropositive hemophiliacs, compared with seronegative hemophiliacs, had significantly decreased lymphoproliferative responses, especially to pokeweed mitogen, tetanus, and Candida stimulations. Immune studies of seven HTLV-III/LAV seropositive hemophiliacs revealed antigen unresponsiveness and decreased T4 cells 2 to 32 months prior to development of full-blown AIDS. Longitudinal immune studies from 1983-85 revealed increasing number of seropositive hemophiliacs with antigen unresponsiveness and decreased T4 cells.

We have designed a closed, inert incubation system for testing the mutagenicity of volatile compounds. The containment properties of this system have been investigated using carbon-14 labelled 1,2-dibromoethane. The recovery of this solvent was about 95% following a 48-h incubation at 37 degrees. Using the Ames Salmonella/microsome mutagenicity assay we have determined the mutagenic potency of 10 common halogenated alkane solvents. Of these 10 compounds, only 1,2-dibromoethane and 1-bromo-2-chloroethane give positive results in the standard test procedure, whereas 7 of the 10 give positive results in the closed system. The specificity observed for reversion of the tester strains and the lack of any significant effect of added rat-liver "S9" fractions suggest that these haloalkanes are direct-acting "base-pair" type mutagens. The mutagenic potencies of the 7 positive compounds range from 0.001 revertants per nanomole for 1,2-dichloroethane to 0.172 revertants per nanomole for 1,2-dibromoethane. A minimum or threshold response level for each material has been calculated.