The effect of hyperglycaemia on haemostasis testing – a volunteer study

We investigated whether the contamination of samples with glucose subsequently tested for haemostasis affected the results, including prothrombin time, activated partial thromboplastin time and fibrinogen concentration. Venous blood was collected from 12 healthy subjects and divided into four aliquots, which were subjected to different degrees of contamination with standard glucose solution (0%, 5%, 10%, 20%). With increasing glucose contamination, prothrombin time increased from mean (SD) 11.0 (0.7) s to 11.2 (0.7) s, 11.5 (0.7) s and 12.2 (0.8) s, all p < 0.001. Activated partial thromboplastin time decreased from 32.3 (0.9) s to 30.9 (0.8) s, 30.8 (0.8) s, and 29.7 (0.7) s, all p < 0.001. Fibrinogen concentration decreased from 3.8 (0.7) g.l^?1 to 3.7 (0.6) g.l^?1, 3.6 (0.6) g.l^?1, and 3.4 (0.6) g.l^?1, all p < 0.001. Bias was clinically meaningful from 5% contamination for activated partial thromboplastin time, 10% contamination for prothrombin time and 20% contamination for fibrinogen concentration. We conclude that if glucose contamination of haemostasis samples is suspected or has occurred, the specimens should not be analysed.     

Ischemic necrosis of the femoral head: An experimental rabbit model

To describe the morphology of the proximal femoral epiphysis in a rabbit model from the ischemic insult to the end of the revascularization process. Ischemia of the femoral head was induced in 32 rabbits at the 8th day of life, by sectioning the joint capsule and the ligamentum teres and dislocating the femoral head. Rabbits were sacrificed at 4, 8, 12, 18, 21, 26, 34, and 48 days after surgery and femoral heads were observed histologically. During the first days following the ischemic injury, large areas underwent necrotic changes. Both epiphyseal and physeal cartilage were thicker than normal and less trabecular bone formation was evident. Bone marrow was also diffusely necrotic within the secondary center of ossification. After day 12th, reparative process started with formation of extensive areas of fibrocartilage and several secondary centers of ossifications. At that stage femoral head deformity was already evident. In the following days the secondary centers of ossification cohalesced and epiphyseal and physeal cartilage resumed a normal appearance, but the femur showed a permanent deformity. In newborn rabbits, the ischemic injury to the femoral head blocked the ossification of the epiphyseal and physeal cartilage associated to necrotic bone marrow within the secondary center of ossification of the femoral head as well as to extensive areas of necrosis of epiphyseal and physeal cartilage. Extensive areas of fibrocartilage and small newly formed ossification centers within the femoral epiphysis were the results of the revascularization process, and femoral head deformity became stable afterward. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:535–541, 2015.     

Identification and characterization of the constituent human serum antibodies elicited by vaccination

Most vaccines confer protection via the elicitation of serum antibodies, yet more than 100 y after the discovery of antibodies, the molecular composition of the human serum antibody repertoire to an antigen remains unknown. Using high-resolution liquid chromatography tandem MS proteomic analyses of serum antibodies coupled with next-generation sequencing of the V gene repertoire in peripheral B cells, we have delineated the human serum IgG and B-cell receptor repertoires following tetanus toxoid (TT) booster vaccination. We show that the TT⁺ serum IgG repertoire comprises ∼100 antibody clonotypes, with three clonotypes accounting for >40% of the response. All 13 recombinant IgGs examined bound to vaccine antigen with K_d ∼ 10⁻⁸–10⁻¹⁰ M. Five of 13 IgGs recognized the same linear epitope on TT, occluding the binding site used by the toxin for cell entry, suggesting a possible explanation for the mechanism of protection conferred by the vaccine. Importantly, only a small fraction (<5%) of peripheral blood plasmablast clonotypes (CD3⁻CD14⁻CD19⁺CD27⁺⁺CD38⁺⁺CD20⁻TT⁺) at the peak of the response (day 7), and an even smaller fraction of memory B cells, were found to encode antibodies that could be detected in the serological memory response 9 mo postvaccination. This suggests that only a small fraction of responding peripheral B cells give rise to the bone marrow long-lived plasma cells responsible for the production of biologically relevant amounts of vaccine-specific antibodies (near or above the K_d). Collectively, our results reveal the nature and dynamics of the serological response to vaccination with direct implications for vaccine design and evaluation.Most approved vaccines confer protection against infectious diseases by the induction of long-lived plasma cells (LLPCs), which secrete antibodies that serve to neutralize and opsonize the pathogen for many years or decades (1–3). Additionally, the generation of memory B cells (mBCs) provides both a mechanism for the rapid synthesis of affinity matured, antigen-specific antibodies following rechallenge and a means to diversify the humoral immune response to confer protection against rapidly evolving viruses or bacteria (4). Although some vaccines elicit antibody titers that remain virtually constant for many decades, for others, including the tetanus toxoid (TT) vaccine, antibody titers wane monotonically over time (5). Booster immunization triggers the rapid expansion and differentiation of cognate B cells, generating antigen-specific plasmablasts that peak in concentration in peripheral blood after 6–7 d and subsequently rapidly decline to nearly undetectable levels (6, 7). Some, but not all, of these peak-wave plasmablasts migrate to specialized niches overwhelmingly located in the bone marrow (BM) and survive as LLPCs (8), which constitute the major source of all classes of Ig in the serum (9).The establishment of serological memory following either primary or booster vaccination is not understood well (10–14). Even though antibody production is the most critical effector function of B-cell immunity, and antigen-specific antibodies in the serum play a key role in protection against pathogen challenge, technical difficulties have precluded direct determination of the identities of the mAbs that comprise the serum antibody response to vaccination. However, recent studies showing that flu vaccination elicits not only neutralizing antibodies but also antibodies that enhance infection by different flu strains (15) underscore the pressing need to develop approaches for delineating the sequences and functionalities of the serum antibodies elicited by vaccination (16).Single-cell cloning has been used to identify neutralizing antibodies encoded by mBCs or plasmablasts in peripheral blood (17). However, although extremely useful for understanding of the structural mechanisms that can lead to the blockade of pathogen infection, the interrogation of single peripheral B cells alone cannot provide information on whether antibodies encoded by single B cells are also produced as secreted IgGs from BM LLPCs, and hence whether they contribute to the serological memory induced by vaccination. A detailed understanding of the diversity of serum antibodies elicited by vaccination, their functionality (e.g., antigen affinity, epitope specificity), and their relative concentrations in the blood can provide key insights toward vaccine evaluation and development.Here, we deployed high-resolution liquid chromatography (LC) tandem MS (MS/MS) (18–20) for the molecular-level analysis of the serum IgG repertoire, combined with deep sequencing of the V gene repertoire of peripheral B lymphocyte subsets (20) and subsequent expression and characterization of representative serum antibodies, to map the dynamics of the human humoral response to vaccination in unprecedented detail. We elected to analyze the response to booster immunization of the TT vaccine because (i) it elicits a highly effective neutralizing response that is protective toward Clostridium tetani challenge; (ii) the vaccine is highly efficacious, and as a result, no deaths from tetanus intoxication have been reported in the United States for individuals who have completed at least primary immunization (21); (iii) TT has been used as a model for analyzing B-cell development following vaccination in humans (6, 22, 23); and (iv) although early serological and mAb studies had pointed to the C-terminal fragment of the toxin heavy chain [recombinant TT fragment C (rTT.C)] as the target for antibody-mediated protection (24), the precise mechanism by which antibodies elicited by the vaccine mediate neutralization has remained unclear.We show that the anti-TT serum IgG repertoire at steady state is composed of a limited number of antibody clonotypes (∼80–100) displaying uniformly high antigen affinity (low nanomolar or subnanomolar), that most of the serum repertoire postboost comprises preexisting (i.e., prevaccination) serum antibody clonotypes, and that there is only partial overlap between the peak-wave plasmablast V gene repertoire and the TT⁺ serum IgG repertoire at steady state after vaccination. We identified several serum monoclonal IgGs that bind to rTT.C, and epitope mapping revealed that all rTT.C-specific antibodies tested bind to an immunodominant linear epitope at the ganglioside-binding site of the toxin that is used for cell entry. Computational antibody docking substantiated that binding of these antibodies to the toxin blocks access to the ganglioside ligand, thus providing a possible mechanistic explanation for how the TT vaccine confers protection. These results highlight the importance of understanding the composition and dynamics of the serum antibody repertoire, together with the V gene repertoire in peripheral B lymphocytes, for the molecular understanding of vaccine function.     

Increased free malondialdehyde concentrations in smokers normalise with a mixed fruit and vegetable juice concentrate: a pilot study.

Background: Cigarette smoking, a cardiovascular risk factor leading to oxygen free radical formation, is involved in the development of serious pathological conditions. On the other hand, a healthy diet and adequate supplementation can help prevent many diseases. The aim of our study was to evaluate in healthy light smokers the effects of supplementation with mixed fruit and vegetable juice powder concentrate on homocysteine metabolism and oxidative status. Methods: In this pilot study, 32 healthy volunteers, 16 light smokers and 16 non-smokers, on twice daily supplementation were monitored at time zero and after 30 days. Plasma homocysteine, and serum vitamin B(12) and folate concentrations were measured by immunoenzymatic assays; reactive oxygen species, total antioxidant capacity and thiol groups by spectrophotometric methods; and total and free malondialdehyde concentrations by gas chromatography-mass spectrometry with isotopic dilution. Results: Baseline free malondialdehyde concentrations were significantly higher in smokers than in non-smokers and normalised after 30-day supplementation. Baseline results for all the other parameters remained unchanged after supplementation, with no significant differences between smokers and non-smokers. Conclusion: This is the first study showing a significant decrease in free malondialdehyde levels in light smokers after 1-month phytonutrient supplementation.     

Valgus extension femoral osteotomy to treat “hinge abduction” in Perthes’ disease

Purpose

“Hinge abduction” is a complication of Perthes’ disease caused by impingement of the extruded superolateral portion of the femoral head against the lateral lip of the acetabulum. Catterall first described femoral valgus extension osteotomy (VGEO) to treat this condition. We report the results of this operation in 16 cases of Perthes’ disease with “hinge abduction”.

Methods

Sixteen hips in 16 patients affected by Perthes’ disease and “hinge abduction” were operated on at a mean age of 10.1 years and followed up an average of 6.5 years later. Before surgery, the mean Iowa hip score was 44.4 points. Preoperative radiographs were taken with the affected hip in maximum adduction in order to calculate the amount of valgus correction. The osteotomy was performed between the greater and the lesser trochanter, and it was fixed with a hip plate.

Results

All the osteotomies healed uneventfully. At follow-up, no patient complained of pain and hip abduction ranged from 20° to 45°. Four out of the 16 patients had a moderate limp, and 12 had an improvement in gait pattern compared to preoperatively. At follow-up, the Iowa hip score totaled a mean of 83.6 points, with a statistically significant improvement in comparison to the preoperative evaluation. At follow-up, two hips were classified as Stulberg II–III, ten hips as Stulberg III, and four as Stulberg IV.

Conclusions

In our hands, VGEO was an effective procedure to treat “hinge abduction” in severe Perthes’ disease with satisfactory results. The main limitation of our study is its short follow-up.     

Detection of Ochratoxin a Using Molecular Beacons and Real-Time PCR Thermal Cycler

We developed a simple and cheap assay for quantitatively detecting ochratoxin A (OTA) in wine. A DNA aptamer available in literature was used as recognition probe in its molecular beacon form, i.e., with a fluorescence-quenching pair at the stem ends. Our aptabeacon could adopt a conformation allowing OTA binding, causing a fluorescence rise due to the increased distance between fluorophore and quencher. We used real-time PCR equipment for capturing the signal. With this assay, under optimized conditions, the entire process can be completed within 1 h. In addition, the proposed system exhibited a good selectivity for OTA against other mycotoxins (ochratoxin B and aflatoxin M1) and limited interference from aflatoxin B1 and patulin. A wide linear detection range (0.2–2000 µM) was achieved, with LOD = 13 nM, r = 0.9952, and R² = 0.9904. The aptabeacon was also applied to detect OTA in red wine spiked with the same dilution series. A linear correlation with a LOD = 19 nM, r = 0.9843, and R² = 0.9708 was observed, with recoveries in the range 63%–105%. Intra- and inter-day assays confirmed its reproducibility. The proposed biosensor, although still being finalized, might significantly facilitate the quantitative detection of OTA in wine samples, thus improving their quality control from a food safety perspective.     

Angiographic aspects of unstable angina

One-hundred and ninety-four patients with unstable angina pectoris (91 "in crescendo" angina and 103 new onset angina) underwent coronary angiography. The angiographic data from both groups were compared in order to discover whether angiographic aspects were related to the various clinical symptoms of coronary artery disease. Patients with recent onset angina had a significant increase (p less than 0.0001) of mono-vessel disease, whereas multi-vessel disease was prevalent in patients with "in crescendo" angina pectoris. Higher prevalence of coronary collaterals was observed in patients with "in crescendo" angina (p less than 0.005). No significant difference was observed in ejection fraction of the two groups. A further analysis was performed in 100 patients with unstable angina pectoris but without prior myocardial infarction (42 "in crescendo" angina and 58 recent onset angina). Also in these patients were found the same results; with the exception of ejection fraction which was more slight in patients with "in crescendo" angina (p less than 0.01). These data confirm that patients with unstable angina are an heterogeneous group in which comparison is unreliable and that the severity of clinical symptoms is not related to the degree of angiographic coronary lesions.     

Ebola viral load at diagnosis associates with patient outcome and outbreak evolution

BACKGROUND. Ebola virus (EBOV) causes periodic outbreaks of life-threatening EBOV disease in Africa. Historically, these outbreaks have been relatively small and geographically contained; however, the magnitude of the EBOV outbreak that began in 2014 in West Africa has been unprecedented. The aim of this study was to describe the viral kinetics of EBOV during this outbreak and identify factors that contribute to outbreak progression.METHODS. From July to December 2014, one laboratory in Sierra Leone processed over 2,700 patient samples for EBOV detection by quantitative PCR (qPCR). Viremia was measured following patient admission. Age, sex, and approximate time of symptom onset were also recorded for each patient. The data was analyzed using various mathematical models to find trends of potential interest.RESULTS. The analysis revealed a significant difference (P = 2.7 × 10^–77) between the initial viremia of survivors (4.02 log₁₀ genome equivalents [GEQ]/ml) and nonsurvivors (6.18 log₁₀ GEQ/ml). At the population level, patient viral loads were higher on average in July than in November, even when accounting for outcome and time since onset of symptoms. This decrease in viral loads temporally correlated with an increase in circulating EBOV-specific IgG antibodies among individuals who were suspected of being infected but shown to be negative for the virus by PCR.CONCLUSIONS. Our results indicate that initial viremia is associated with outcome of the individual and outbreak duration; therefore, care must be taken in planning clinical trials and interventions. Additional research in virus adaptation and the impacts of host factors on EBOV transmission and pathogenesis is needed.     

The role of social media in clubfoot: information sharing and social support

PurposeClubfoot is the most common congenital foot deformity in children. Caregivers often seek medical information on the internet. The aim of the study was to characterize how social media is used by caregivers to access medical information.MethodsA search was performed on Facebook, Twitter and YouTube platforms. Information was quantitatively assessed. Comments were qualitatively assessed, and the Kruskal-Wallis test was used to study thematic comment distribution.ResultsIn total, 58 Facebook groups and pages, 109 YouTube accounts and ten Twitter accounts related to clubfoot were discovered from 2007 to 2019. Facebook groups and pages had a collective 56 123 members and 80 544 total likes, respectively. YouTube had a collective 3 280 454 views, with 54 969 total comments throughout the accounts. Comment themes most commonly included sharing information and advice (38.7%), appreciation and success stories (12.8%), emotional support (12.7%) and social media as a second opinion (11.9%). Facebook groups contained a significantly higher number of comments related to ‘social media as a second opinion’ compared with Facebook pages (p = 0.001), Twitter (p = 0.016) and YouTube (p < 0.0001) while YouTube contained a significantly lower number of comments related to ‘sharing information’ compared with Facebook groups, pages and Twitter (p < 0.0001).ConclusionSocial media continues to be a growing tool for information sharing and the findings of this study highlight the importance placed by caregivers on the advice of their peers. The online presence of caregivers may represent an opportunity for orthopaedic surgeons to communicate with patients and help them make informed decisions.Level of evidenceIV