A new aspect of in vitro antimicrobial leukocyte- and platelet-rich plasma activity based on flow cytometry assessment

The current literature suggests that the antibacterial effect of leukocyte- and platelet-rich plasma (L-PRP) is directly related to platelet and leukocyte concentrations. The aim of this study was twofold: first, to evaluate the antimicrobial effect of L-PRP against selected bacterial strains in vitro, and second, to correlate this effect with leukocyte and platelet content in the final concentration. Blood was collected from 20 healthy males, and L-PRP, acellular plasma (AP), and autologous thrombin were consecutively prepared. Flow cytometry analysis of the blood, L-PRP, and AP was performed. The L-PRP gel, liquid L-PRP, and thrombin samples were tested in vitro for their antibacterial properties against seven selected bacterial strains using the Kirby–Bauer disk-diffusion method. There was notable antimicrobial activity against selected bacterial strains. No statistically significant correlations between antimicrobial activities and the platelet concentration in L-PRP were observed. Statistically significant positive correlations between selected leukocyte subtypes and antimicrobial activity were noted. A negative correlation was found between elevated monocyte count and antimicrobial activity of L-PRP against one bacterial strain studied. L-PRP possesses antimicrobial activity and can be potentially useful in the fight against certain postoperative infections. The bactericidal effect of L-PRP is caused by leukocytes, and there exists a relationship among selected leukocyte subtypes and L-PRP antimicrobial activity.     

Does damage of perivascular astrocytes in multiple sclerosis plaques participate in blood-brain barrier permeability?

In six young and two senile MS cases perivascular astrocytes within demyelination lesions were evaluated immunocytochemically. The peroxidase-antiperoxidase method of Sternberger et al. (1970) was used for visualization of glial fibrillary acidic protein (GFAP). In all cases very weak immunoreactivity of perivascular astrocytes was noted. Accumulation of perivascular glial fibers, and infrequently their fragmentation were observed both within active and old demyelination plaques. Clasmatodendrosis, Rosenthal's fibers and prominent regressive changes of astrocyte perikarya were found only in old plaques. A lack of immunoreactivity of perivascular astrocytes was noted within old demyelination lesions. The background of the latter was often immunonegative to GFAP. It is suggested that secondary damage of perivascular astrocytes influences vascular permeability within demyelination lesions including old plaques.     

Association of tumor necrosis factor and human leukocyte antigen DRB1 alleles with Graves' ophthalmopathy

Tumor necrosis factor (TNF)-alpha plays a central role in the development of ophthalmopathy in patients with Graves' disease (GD). The aim of this study was to investigate the association of TNF promoter polymorphisms at positions -1031 (T-1031C), -863 (C-863A), -857 (C-857T), -308 (G-308A), and -238 (G-238A) with Graves' ophthalmopathy (GO). We studied the distribution of TNF and human leukocyte antigen (HLA) DRB1 alleles in 228 Polish white patients with GD, 106 of whom had ophthalmopathy (NOSPECS class > or = III) and 248 healthy subjects. TNF -308A and HLA-DRB1*03 alleles were significantly increased in patients with GD compared with healthy subjects. Stratification analysis revealed no independent association of -308A with GD when the DRB1*03 status was considered. Subdividing GD according to eye involvement revealed that the distribution of TNF promoter haplotypes differed significantly in patients with or without ophthalmopathy. The haplotype containing the -238A allele was absent in GO. The association of G-238A with GO was independent of DRB1 alleles. These results indicate that TNF G-308A is associated with susceptibility to GD (however, this association is not independent of HLA-DRB1*03) and that TNF G-238A is associated with the development of ophthalmopathy, suggesting that G-238A or a gene in linkage disequilibrium may be disease modifying in GD.     

Dysferlin mutations in LGMD2B, Miyoshi myopathy, and atypical dysferlinopathies

DYSF encoding dysferlin is mutated in Miyoshi myopathy and Limb-Girdle Muscular Dystrophy type 2B, the two main phenotypes recognized in dysferlinopathies. Dysferlin deficiency in muscle is the most relevant feature for the diagnosis of dysferlinopathy and prompts the search for mutations in DYSF. DYSF, located on chromosome 2p13, contains 55 coding exons and spans 150 kb of genomic DNA. We performed a genomic analysis of the DYSF coding sequence in 34 unrelated patients from various ethnic origins. All patients showed an absence or drastic decrease of dysferlin expression in muscle. A primary screening of DYSF using SSCP or dHPLC of PCR products of each of 55 exons of the gene was followed by sequencing whenever a sequence variation was detected. All together, 54 sequence variations were identified in DYSF, 50 of which predicting either a truncated protein or one amino-acid substitution and most of them (34 out of 54) being novel. In 23 patients, we identified two pathogenic mutations, while only one was identified in 11 patients. These mutations were widely spread in the coding sequence of the gene without any mutational "hotspot."     

Comparison of leukocyte populations from bronchoalveolar lavage and induced sputum in the evaluation of cellular composition and nitric oxide production in patients with bronchial asthma

Bronchoalveolar lavage (BAL) or induced sputum (IS) techniques may provide leukocytes for the evaluation of airway inflammatory response in bronchial asthma. The aim of the present study was to compare features of leukocyte populations obtained by the two different methods regarding the cell types and their activity in patients with bronchial asthma. The nitric oxide (NO) level released from the cells was measured as a marker of their activity. Pulmonary leukocytes were obtained from the BAL and IS of 11 asthmatic patients in stable condition at the time of the study. The BAL and IS leukocyte populations varied in cell count and NO production. Macrophages were the predominant leukocyte population in BAL (median (Me) = 83.0%, range 67.9-88.4%), whereas sputum sediments were found to consist mainly of neutrophils (Me = 55.7%, range 29.0-64.9%). The IS leukocytes released much more NO (p = 0.0022) than the BAL leukocytes. In spite of these quantitative differences, a similar pattern of NO production was observed in BAL and in IS cells. Both BAL and IS leukocyte populations produced almost the same amounts of NO before and after lipopolysaccharide stimulation (p = 0.9063, p = 0.4801, respectively). Furthermore, a slight positive correlation Spearman's rank (RS) = 0.5578, p = 0.0594) was noticed between the neutrophil percentages and NO levels produced by BAL cells, whereas in IS a statistically significant correlation between the percentage of neutrophils and the levels of NO (RS = 0.6643, p = 0.0184) was observed. In conclusion, the BAL and IS leukocyte populations are different in cell type, their size and activity. Depending on the asthma severity and the type of cells needed in a study, either BAL or IS specimens may be chosen as a source of pulmonary leukocytes. The use of IS as a noninvasive technique is supposed to be potential value particularly in the study of the airway inflammatory response mediated mainly by neutrophils, i.e. during and/or after exacerbation of the disease. Based on our results, a possible contribution of neutrophils in the production of NO in the airways of asthmatic patients can be proposed apart from other cells such as macrophages.     

Oxidative modification of type II collagen differentially affects its arthritogenic and tolerogenic capacity in experimental arthritis

INTRODUCTION: Oxidative modification of proteins affects their biological properties. Previously we have shown that hypochlorite (HOCl), the product of activated neutrophils, enhances protein immunogenecity. Collagen type II, a primary component of cartilage, is commonly used in the induction of arthritis in animals (CIA). The aim of this study was to examine whether HOCl may affect immunogenic, tolerogenic, and arthritogenic properties of collagen. MATERIALS AND METHODS: DBA/J mice were injected with either native (CNAT) or chlorinated collagen (CHOCl) to induce arthritis. The effect of chlorination on collagen properties was measured by evaluation of incidence and severity of CIA. Moreover, the concentration of serum anti-collagen IgG antibodies and myeloperoxidase (MPO) activity in inflamed joints was determined. RESULTS: Mice immunized with CNAT in adjuvant developed arthritis (CIA) with an incidence of 69%. CNAT also exerted tolerogenic properties when injected intravenously either before or shortly after primary immunization, resulting in decreased incidence and severity of CIA, reduced MPO activity in inflamed joints, and lowered serum levels of anti-CNAT IgG anti-bodies. Chlorination of collagen significantly diminished its ability to induce CIA and to trigger generation of anti-CNAT IgG antibodies. Interestingly, chlorination did not affect tolerogenic properties of collagen administered prior to primary immunization with CNAT. CONCLUSIONS: These results suggest that chlorination of collagen may selectively affect functional epitopes of collagen. It is likely that in inflamed joints, neutrophil derived HOCl, in some circumstances, will destroy arthritogenic and immunogenic B cell epitopes, while regulatory T cell epitopes will be preserved.     

How Do Medical Students Perceive Diversity in Orthopaedic Surgery,and How Do Their Perceptions Change After an Orthopaedic Clinical Rotation?

BackgroundA diverse physician workforce improves the quality of care for all patients, and there is a need for greater diversity in orthopaedic surgery. It is important that medical students of diverse backgrounds be encouraged to pursue the specialty, but to do so, we must understand students’ perceptions of diversity and inclusion in orthopaedics. We also currently lack knowledge about how participation in an orthopaedic clinical rotation might influence these perceptions.Questions/purposes(1) How do the perceptions of diversity and inclusion in orthopaedic surgery compare among medical students of different gender identities, races or ethnicities, and sexual orientations? (2) How do perceptions change after an orthopaedic clinical rotation among members of demographic groups who are not the majority in orthopaedics (that is, cis-gender women, underrepresented racial minorities, other racial minorities, and nonheterosexual people)?MethodsWe surveyed students from 27 US medical schools who had completed orthopaedic rotations. We asked about their demographic characteristics, rotation experience, perceptions of diversity and inclusion in orthopaedics, and personal views on specialty choice. Questions were derived from diversity, equity, and inclusion climate surveys used at major academic institutions. Cis-gender men and cis-gender women were defined as those who self-identified their gender as men or women, respectively, and were not transgender. Forty-five percent (59 of 131) of respondents were cis-men and 53% (70 of 131) were cis-women; 49% (64 of 131) were white, 20% (26 of 131) were of underrepresented racial minorities, and 31% (41 of 131) were of other races. Eighty-five percent (112 of 131) of respondents were heterosexual and 15% (19 of 131) reported having another sexual orientation. We compared prerotation and postrotation perceptions of diversity and inclusion between majority and nonmajority demographic groups for each demographic domain (for example, cis-men versus cis-women). We also compared prerotation to postrotation perceptions within each nonmajority demographic group. To identify potential confounding variables, we performed univariate analysis to compare student and rotation characteristics across the demographic groups, assessed using an alpha of 0.05. No potential confounders were identified. Statistical significance was assessed at a Bonferroni-adjusted alpha of 0.0125. Our estimated response percentage was 26%. To determine limitations of nonresponse bias, we compared all early versus late responders and found that for three survey questions, late responders had a more favorable perception of diversity in orthopaedic surgery, whereas for most questions, there was no difference.ResultsBefore rotation, cis-women had lower agreement that diversity and inclusion are part of orthopaedic culture (mean score 0.96 ± 0.75) compared with cis-men (1.4 ± 1.1) (mean difference 0.48 [95% confidence interval 0.16 to 0.81]; p = 0.004), viewed orthopaedic surgery as less diverse (cis-women 0.71 ± 0.73 versus cis-men 1.2 ± 0.92; mean difference 0.49 [95% CI 0.20 to 0.78]; p = 0.001) and more sexist (cis-women 1.3 ± 0.92 versus cis-men 1.9 ± 1.2; mean difference 0.61 [95% CI 0.23 to 0.99]; p = 0.002), believed they would have to work harder than others to be valued equally (cis-women 2.8 ± 1.0 versus cis-men 1.9 ± 1.3; mean difference 0.87 [95% CI 0.45 to 1.3]; p < 0.001), and were less likely to pursue orthopaedic surgery (cis-women 1.4 ± 1.4 versus cis-men 2.6 ± 1.1; mean difference 1.2 [95% CI 0.76 to 1.6]; p < 0.001). Before rotation, underrepresented minorities had less agreement that diversity and inclusion are part of orthopaedic surgery culture (0.73 ± 0.72) compared with white students (1.5 ± 0.97) (mean difference 0.72 [95% CI 0.35 to 1.1]; p < 0.001). Many of these differences between nonmajority and majority demographic groups ceased to exist after rotation. Compared with their own prerotation beliefs, after rotation, cis-women believed more that diversity and inclusion are part of orthopaedic surgery culture (prerotation mean score 0.96 ± 0.75 versus postrotation mean score 1.2 ± 0.96; mean difference 0.60 [95% CI 0.22 to 0.98]; p = 0.002) and that orthopaedic surgery is friendlier (prerotation 2.3 ± 1.2 versus postrotation 2.6 ± 1.1; mean difference 0.41 [95% CI 0.14 to 0.69]; p = 0.004), more diverse (prerotation 0.71 ± 0.73 versus postrotation 1.0 ± 0.89; mean difference 0.28 [95% CI 0.08 to 0.49]; p = 0.007), less sexist (prerotation 1.3 ± 0.92 versus postrotation 1.9 ± 1.0; mean difference 0.63 [95% CI 0.40 to 0.85]; p < 0.001), less homophobic (prerotation 2.1 ± 1.0 versus postrotation 2.4 ± 0.97; mean difference 0.27 [95% CI 0.062 to 0.47]; p = 0.011), and less racist (prerotation 2.3 ± 1.1 versus postrotation 2.5 ± 1.1; mean difference 0.28 [95% CI 0.099 to 0.47]; p = 0.003). Compared with before rotation, after rotation cis-women believed less that they would have to work harder than others to be valued equally on the rotation (prerotation 2.8 ± 1.0 versus postrotation 2.5 ± 1.0; mean difference 0.31 [95% CI 0.12 to 0.50]; p = 0.002), as did nonheterosexual students (prerotation 2.4 ± 1.4 versus postrotation 1.8 ± 1.3; mean difference 0.56 [95% 0.21 to 0.91]; p = 0.004). Underrepresented minority students saw orthopaedic surgery as less sexist after rotation compared with before rotation (prerotation 1.5 ± 1.1 versus postrotation 2.0 ± 1.1; mean difference 0.52 [95% CI 0.16 to 0.89]; p = 0.007).ConclusionEven with an estimated 26% response percentage, we found that medical students of demographic backgrounds who are not the majority in orthopaedics generally perceived that orthopaedic surgery is less diverse and inclusive than do their counterparts in majority groups, but these views often change after a clinical orthopaedic rotation.Clinical RelevanceThese perceptions may be a barrier to diversification of the pool of medical student applicants to orthopaedics. However, participation in an orthopaedic surgery rotation is associated with mitigation of many of these negative perceptions among diverse students. Medical schools have a responsibility to develop a diverse workforce, and given our findings, schools should promote participation in a clinical orthopaedic rotation. Residency programs and orthopaedic organizations can also increase exposure to the field through the rotation and other means. Doing so may ultimately diversify the orthopaedic surgeon workforce and improve care for all orthopaedic patients.     

Identification of a new familial case of 3q29 deletion syndrome associated with cleft lip and palate via whole-exome sequencing

Many unbalanced large copy number variants reviewed in the paper are associated with syndromic orofacial clefts, including a 1.6 Mb deletion on chromosome 3q29. The current report presents a new family with this recurrent deletion identified via whole-exome sequencing and confirmed by array comparative genomic hybridization. The proband exhibited a more severe clinical phenotype than his affected mother, comprising right-sided cleft lip/alveolus and cleft palate, advanced dental caries, heart defect, hypospadias, psychomotor, and speech delay, and an intellectual disability. Data analysis from the 3q29 registry revealed that the 3q29 deletion increases the risk of clefting by nearly 30-fold. No additional rare and pathogenic nucleotide variants were identified that could explain the clefting phenotype and observed intrafamilial phenotypic heterogeneity. These data suggest that the 3q29 deletion may be the primary risk factor for clefting, with additional genomic variants located outside the coding sequences, methylation changes, or environmental exposure serving as modifiers of this risk. Additional studies, including whole-genome sequencing or methylation analyses, should be performed to identify genetic factors underlying the phenotypic variation associated with the recurrent 3q29 deletion.