Optimization and Validation of FePro Cell Labeling Method

Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12–16 hours) incubation time and uses relatively high dose of Pro (5–6 µg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 µg/ml and Pro 0.75 to 3 µg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached ∼30–35 pg-iron/cell at 24 h when labeled with 100 µg/ml of Fe and 3 µg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved ∼10 pg-iron/cell at 48 h when labeled with 100 µg/ml of Fe and 3 µg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17.2) were labeled. Labeling for 4 hours using 100 µg/ml of Fe and 3 µg/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability. The new technique with short incubation time using 100 µg/ml of Fe and 3 µg/ml of Pro is effective in labeling cells for cellular MRI.     

Facilitating the Recruitment of Minority Ethnic People into Research: Qualitative Case Study of South Asians and Asthma

Background

There is international interest in enhancing recruitment of minority ethnic people into research, particularly in disease areas with substantial ethnic inequalities. A recent systematic review and meta-analysis found that UK South Asians are at three times increased risk of hospitalisation for asthma when compared to white Europeans. US asthma trials are far more likely to report enrolling minority ethnic people into studies than those conducted in Europe. We investigated approaches to bolster recruitment of South Asians into UK asthma studies through qualitative research with US and UK researchers, and UK community leaders.

Methods and Findings

Interviews were conducted with 36 researchers (19 UK and 17 US) from diverse disciplinary backgrounds and ten community leaders from a range of ethnic, religious, and linguistic backgrounds, followed by self-completion questionnaires. Interviews were digitally recorded, translated where necessary, and transcribed. The Framework approach was used for analysis. Barriers to ethnic minority participation revolved around five key themes: (i) researchers'' own attitudes, which ranged from empathy to antipathy to (in a minority of cases) misgivings about the scientific importance of the question under study; (ii) stereotypes and prejudices about the difficulties in engaging with minority ethnic populations; (iii) the logistical challenges posed by language, cultural differences, and research costs set against the need to demonstrate value for money; (iv) the unique contexts of the two countries; and (v) poorly developed understanding amongst some minority ethnic leaders of what research entails and aims to achieve. US researchers were considerably more positive than their UK counterparts about the importance and logistics of including ethnic minorities, which appeared to a large extent to reflect the longer-term impact of the National Institutes of Health''s requirement to include minority ethnic people.

Conclusions

Most researchers and community leaders view the broadening of participation in research as important and are reasonably optimistic about the feasibility of recruiting South Asians into asthma studies provided that the barriers can be overcome. Suggested strategies for improving recruitment in the UK included a considerably improved support structure to provide academics with essential contextual information (e.g., languages of particular importance and contact with local gatekeepers), and the need to ensure that care is taken to engage with the minority ethnic communities in ways that are both culturally appropriate and sustainable; ensuring reciprocal benefits was seen as one key way of avoiding gatekeeper fatigue. Although voluntary measures to encourage researchers may have some impact, greater impact might be achieved if UK funding bodies followed the lead of the US National Institutes of Health requiring recruitment of ethnic minorities. Such a move is, however, likely in the short- to medium-term, to prove unpopular with many UK academics because of the added “hassle” factor in engaging with more diverse populations than many have hitherto been accustomed to. Please see later in the article for the Editors'' Summary     

Preserved MHC Class II Antigen Processing in Monocytes from HIV-Infected Individuals

Background

MHC-II restricted CD4+ T cells are dependent on antigen presenting cells (APC) for their activation. APC dysfunction in HIV-infected individuals could accelerate or exacerbate CD4+ T cell dysfunction and may contribute to increased levels of immunodeficiency seen in some patients regardless of their CD4+ T cell numbers. Here we test the hypothesis that APC from HIV-infected individuals have diminished antigen processing and presentation capacity.

Methodology/Principal Findings

Monocytes (MN) were purified by immuno-magnetic bead isolation techniques from HLA-DR1.01+ or DR15.01+ HIV-infected and uninfected individuals. MN were analyzed for surface MHC-II expression and for antigen processing and presentation capacity after overnight incubation with soluble antigen or peptide and HLA-DR matched T cell hybridomas. Surface expression of HLA-DR was 20% reduced (p<0.03) on MN from HIV-infected individuals. In spite of this, there was no significant difference in antigen processing and presentation by MN from 14 HIV-infected donors (8 HLA-DR1.01+ and 6 HLA-DR15.01+) compared to 24 HIV-uninfected HLA-matched subjects.

Conclusions/Significance

We demonstrated that MHC class II antigen processing and presentation is preserved in MN from HIV-infected individuals. This further supports the concept that this aspect of APC function does not further contribute to CD4+ T cell dysfunction in HIV disease.     

A preliminary biomechanical assessment of a polymer composite hip implant using an infrared thermography technique validated by strain gage measurements

With the resurgence of composite materials in orthopaedic applications, a rigorous assessment of stress is needed to predict any failure of bone-implant systems. For current biomechanics research, strain gage measurements are employed to experimentally validate finite element models, which then characterize stress in the bone and implant. Our preliminary study experimentally validates a relatively new nondestructive testing technique for orthopaedic implants. Lock-in infrared (IR) thermography validated with strain gage measurements was used to investigate the stress and strain patterns in a novel composite hip implant made of carbon fiber reinforced polyamide 12 (CF/PA12). The hip implant was instrumented with strain gages and mechanically tested using average axial cyclic forces of 840 N, 1500 N, and 2100 N with the implant at an adduction angle of 15 deg to simulate the single-legged stance phase of walking gait. Three-dimensional surface stress maps were also obtained using an IR thermography camera. Results showed almost perfect agreement of IR thermography versus strain gage data with a Pearson correlation of R(2) = 0.96 and a slope = 1.01 for the line of best fit. IR thermography detected hip implant peak stresses on the inferior-medial side just distal to the neck region of 31.14 MPa (at 840 N), 72.16 MPa (at 1500 N), and 119.86 MPa (at 2100 N). There was strong correlation between IR thermography-measured stresses and force application level at key locations on the implant along the medial (R(2) = 0.99) and lateral (R(2) = 0.83 to 0.99) surface, as well as at the peak stress point (R(2) = 0.81 to 0.97). This is the first study to experimentally validate and demonstrate the use of lock-in IR thermography to obtain three-dimensional stress fields of an orthopaedic device manufactured from a composite material.     

Prospective survey of veterinary practitioners’ primary assessment of equine colic: clinical features,diagnoses, and treatment of 120 cases of large colon impaction

Background

Large colon impactions are a common cause of colic in the horse. There are no scientific reports on the clinical presentation, diagnostic tests and treatments used in first opinion practice for large colon impaction cases. The aim of this study was to describe the presentation, diagnostic approach and treatment at the primary assessment of horses with large colon impactions.

Methods

Data were collected prospectively from veterinary practitioners on the primary assessment of equine colic cases over a 12 month period. Inclusion criteria were a diagnosis of primary large colon impaction and positive findings on rectal examination. Data recorded for each case included history, signalment, clinical and diagnostic findings, treatment on primary assessment and final case outcome. Case outcomes were categorised into three groups: simple medical (resolved with single treatment), complicated medical (resolved with multiple medical treatments) and critical (required surgery, were euthanased or died). Univariable analysis using one-way ANOVA and Tukey’s post-hoc test, Kruskal Wallis with Dunn’s post-hoc test and Chi squared analysis were used to compare between different outcome categories.

Results

1032 colic cases were submitted by veterinary practitioners: 120 cases met the inclusion criteria for large colon impaction. Fifty three percent of cases were categorised as simple medical, 36.6% as complicated medical, and 9.2% as critical. Most cases (42.1%) occurred during the winter. Fifty nine percent of horses had had a recent change in management, 43% of horses were not ridden, and 12.5% had a recent / current musculoskeletal injury. Mean heart rate was 43bpm (range 26-88) and most cases showed mild signs of pain (67.5%) and reduced gut sounds (76%). Heart rate was significantly increased and gut sounds significantly decreased in critical compared to simple medical cases (p<0.05). Fifty different treatment combinations were used, with NSAIDs (93%) and oral fluids (71%) being administered most often.

Conclusions

Large colon impactions typically presented with mild signs of colic; heart rate and gut sounds were the most useful parameters to distinguish between simple and critical cases at the primary assessment. The findings of seasonal incidence and associated management factors are consistent with other studies. Veterinary practitioners currently use a wide range of different treatment combinations for large colon impactions.

     

Evolutionary History of the PER3 Variable Number of Tandem Repeats (VNTR): Idiosyncratic Aspect of Primate Molecular Circadian Clock

The PER3 gene is one of the clock genes, which function in the core mammalian molecular circadian system. A variable number of tandem repeats (VNTR) locus in the 18th exon of this gene has been strongly associated to circadian rhythm phenotypes and sleep organization in humans, but it has not been identified in other mammals except primates. To better understand the evolution and the placement of the PER3 VNTR in a phylogenetical context, the present study enlarges the investigation about the presence and the structure of this variable region in a large sample of primate species and other mammals. The analysis of the results has revealed that the PER3 VNTR occurs exclusively in simiiforme primates and that the number of copies of the primitive unit ranges from 2 to 11 across different primate species. Two transposable elements surrounding the 18th exon of PER3 were found in primates with published genome sequences, including the tarsiiforme Tarsius syrichta, which lacks the VNTR. These results suggest that this VNTR may have evolved in a common ancestor of the simiiforme branch and that the evolutionary copy number differentiation of this VNTR may be associated with primate simiiformes sleep and circadian phenotype patterns.     

Prevalence of Cryptosporidium-Associated Diarrhea in a High Altitude-Community of Saudi Arabia Detected by Conventional and Molecular Methods

Cryptosporidium diarrhea represents a relevant clinical problem in developing countries. In Al-Taif, a city of Saudi Arabia that lies at an altitude of an around 2 km above the sea level, Cryptosporidium infection seems to be undiagnosed in nearly all clinical laboratories. Furthermore, nothing was published regarding Cryptosporidium-associated diarrhea in this area. The objectives of this research were to (1) determine the Cryptosporidium prevalence among patients with diarrhea and (2) to estimate the performances of 3 different diagnostic methods. Total 180 diarrheal fecal samples, 1 sample per patient, were collected between January and August 2013. Samples were screened for Cryptosporidium with modified Zeihl Neelsen (ZN) microscopy, RIDA® Quick lateral flow (LF) immunotest, and a previously published PCR. The Cryptosporidium prevalence rate was 9.4% (17/180), 10% (18/180), and 11.6% (21/180) by microscopy, LF, and PCR test, respectively. Infection was significantly (P=0.004) predominant among children <5 years (22%) followed by children 5-9 years (11.1%). Although infection was higher in males than in females (16.2% males and 8.5% females), the difference was not statistically significant (P=0.11). Compared to PCR, the sensitivity of microscopy and the LF test were 80.9%, 85.7%, respectively. To conclude, high Cryptosporidium-associated diarrhea was found in this area especially in children ≤9 years. The PCR test showed the best performance followed by the LF test and ZN staining microscopy. The primary health care providers in Al-Taif need to be aware of and do testing for this protozoon, particularly for children seen with diarrhea.