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.     

Heterozygous germline BLM mutations increase susceptibility to asbestos and mesothelioma

Rare biallelic BLM gene mutations cause Bloom syndrome. Whether BLM heterozygous germline mutations (BLM^+/−) cause human cancer remains unclear. We sequenced the germline DNA of 155 mesothelioma patients (33 familial and 122 sporadic). We found 2 deleterious germline BLM^+/− mutations within 2 of 33 families with multiple cases of mesothelioma, one from Turkey (c.569_570del; p.R191Kfs*4) and one from the United States (c.968A>G; p.K323R). Some of the relatives who inherited these mutations developed mesothelioma, while none with nonmutated BLM were affected. Furthermore, among 122 patients with sporadic mesothelioma treated at the US National Cancer Institute, 5 carried pathogenic germline BLM^+/− mutations. Therefore, 7 of 155 apparently unrelated mesothelioma patients carried BLM^+/− mutations, significantly higher (P = 6.7E-10) than the expected frequency in a general, unrelated population from the gnomAD database, and 2 of 7 carried the same missense pathogenic mutation c.968A>G (P = 0.0017 given a 0.00039 allele frequency). Experiments in primary mesothelial cells from Blm^+/− mice and in primary human mesothelial cells in which we silenced BLM revealed that reduced BLM levels promote genomic instability while protecting from cell death and promoted TNF-α release. Blm^+/− mice injected intraperitoneally with asbestos had higher levels of proinflammatory M1 macrophages and of TNF-α, IL-1β, IL-3, IL-10, and IL-12 in the peritoneal lavage, findings linked to asbestos carcinogenesis. Blm^+/− mice exposed to asbestos had a significantly shorter survival and higher incidence of mesothelioma compared to controls. We propose that germline BLM^+/− mutations increase the susceptibility to asbestos carcinogenesis, enhancing the risk of developing mesothelioma.

In the United States, the incidence rate of mesothelioma varies between fewer than one case per 100,000 persons in states with no asbestos industry to two to three cases per 100,000 persons in states with an asbestos industry (1, 2). Asbestos causes DNA damage and apoptosis (3) and promotes a chronic inflammatory reaction that supports the emergence of malignant cells (4). Fortunately, only a small fraction of exposed individuals develop mesothelioma; for example, 4.6% of deaths in miners who worked in asbestos mines for over 10 y were caused by mesothelioma (1). Therefore, multiple cases of mesothelioma in the same family are rare and suggest genetic predisposition (5). In 2001, we discovered that susceptibility to mesothelioma was transmitted in a Mendelian fashion across multiple generations in some Turkish families exposed to the carcinogenic fiber erionite, pointing to gene × environment interaction (G×E) as the cause (6). In 2011, we discovered that carriers of heterozygous germline BRCA1-associated protein–1 (BAP1) mutations (BAP1^+/−) developed mesothelioma and uveal melanoma (5), findings expanded and confirmed by us and by multiple research teams (reviewed in refs. 1, 7, 8). Moreover, heterozygous germline Bap1 mutations (Bap1^+/−) significantly increased susceptibility to asbestos-induced mesothelioma in mice (9, 10), evidence of G×E. Reduced BAP1 levels impair DNA repair (11) as well as different forms of cell death (3, 12) and induce metabolic alterations (13–15) that together favor cancer development and growth.Recent studies revealed that mesothelioma may also develop among carriers of germline mutations of additional tumor-suppressor genes that cause well-defined cancer syndromes, including MLH1 and MLH3 (Lynch syndrome), TP53 (Li–Fraumeni syndrome), and BRCA1-2 (Breast and Ovarian Cancer syndrome) (16, 17). When all germline mutations are combined, it has been estimated that about 12% of mesotheliomas occur in carriers of heterozygous germline mutations of BAP1, the most frequent mutation among patients with mesothelioma, or of other tumor suppressors. Some of these mutations may sensitize the host to asbestos carcinogenesis, according to a G×E scenario (17). Thus, presently, mesothelioma is considered an ideal model to study G×E in cancer (17). As part of the Healthy Nevada Project (HNP), we are studying G×E in northern Nevada, a region with an unusually high risk of exposure to carcinogenic minerals and arsenic, which may be related to the high cancer rates in this region (18). We are investigating genetic variants that may increase cancer risk upon exposure to carcinogens to implement preventive strategies.Biallelic mutations of the Bloom syndrome gene (BLM) cause Bloom syndrome, an autosomal-recessive tumor predisposition syndrome characterized by pre- and postnatal growth deficiency, photosensitivity, type 2 diabetes, and greatly increased risk of developing various types of cancers. BLM is a RecQ helicase enzyme that modulates DNA replication and repair of DNA damage by homologous recombination (19). In patients affected by Bloom syndrome, the absence of the BLM protein causes chromosomal instability, increased number of sister chromatid exchanges, and increased numbers of micronuclei (20–22). In addition, BLM is required for p53-mediated apoptosis (23), a process critical to eliminate cells that have accumulated DNA damage. Impaired DNA repair together with altered apoptosis resulted in increased cancer incidence (17, 24). Of course, inactivating germline BLM heterozygous (BLM^+/−) mutations are much more common than biallelic BLM (BLM^−/−) mutations, with an estimated frequency in the general population of 1 in 900 based on data from the Exome Aggregation Consortium (25). BLM^+/− mutation carriers do not show an obvious phenotype; however, some studies have suggested that carriers of these mutations may have an increased cancer risk (17, 24). Mice carrying Blm^+/− mutations are prone to develop a higher rate of malignancies in the presence of contributing factors, such as concurrent heterozygous mutations of the adenomatous polyposis coli (Apc) gene, or upon infection with murine leukemia virus (26). However, in studies in which Blm^+/− mice were crossed with tuberous sclerosis 1-deficient (Tsc1^+/−) mice that are predisposed to renal cystadenomas and carcinomas, Wilson et al. found that Tsc1^+/− Blm^+/− mice did not show significantly more renal cell carcinomas compared with Tsc1^+/− Blm^WT mice (27). In humans, a large study involving 1,244 patients with colon cancer and 1,839 controls of Ashkenazi Jewish ancestry, in which BLM^+/− frequency is as high as 1 in 100 individuals (28), suggested that carriers of germline BLM^+/− mutations might have a twofold increase in colorectal cancer (CRC) (29). A smaller study did not confirm these results, but reported a trend of increasing incidence of adenomas—premalignant lesions—among BLM^+/− mutation carriers (30). In addition, BLM^+/− mutations were found overrepresented among early-onset (<45 y old) CRC patients (25). Other studies associated BLM^+/− mutations to an increased risk of breast (31, 32) and prostate cancer (33), but the low power of these studies hampered definite conclusions. In summary, it appears possible that BLM^+/− mutations may increase cancer risk in the presence of contributing factors.     

Effects of Azithromycin,Metronidazole, Amoxicillin,and Metronidazole plus Amoxicillin on an In Vitro Polymicrobial Subgingival Biofilm Model

Chronic periodontitis is one of the most prevalent human diseases and is caused by dysbiosis of the subgingival microbiota. Treatment involves primarily mechanical disruption of subgingival biofilms and, in certain cases, adjunctive use of systemic antibiotic therapy. In vitro biofilm models have been developed to study antimicrobial agents targeting subgingival species. However, these models accommodate a limited number of taxa, lack reproducibility, and have low throughput. We aimed to develop an in vitro multispecies biofilm model that mimics subgingival plaque, to test antimicrobial agents. Biofilms were cultivated using the Calgary Biofilm Device and were exposed to amoxicillin (AMX), metronidazole (MTZ), azithromycin (AZM), and AMX-MTZ at four different concentrations for 12, 24, or 36 h. Chlorhexidine (CHX) (0.12%) was used as the positive control. The compositions of the biofilms were analyzed by checkerboard DNA-DNA hybridization, and the percent reduction in biofilm metabolic activity was determined using 2,3,5-triphenyltetrazolium chloride and spectrophotometry. Thirty-five of the 40 species used in the inoculum were consistently recovered from the resulting in vitro biofilms. After 36 h of exposure at the 1:27 dilution, AMX-MTZ reduced metabolic activity 11% less than CHX (q = 0.0207) but 54% more than AMX (q = 0.0031), 72% more than MTZ (q = 0.0031), and 67% more than AZM (q = 0.0008). Preliminary evidence of a synergistic interaction between AMX and MTZ was also observed. In summary, we developed reproducible biofilms with 35 subgingival bacterial species, and our results suggested that the combination of AMX and MTZ had greater antimicrobial effects on these in vitro multispecies biofilms than expected on the basis of the independent effects of the drugs.     

Immunophenotypic features by multiparameter flow cytometry can help distinguish low grade B‐cell lymphomas with plasmacytic differentiation from plasma cell proliferative disorders with an unrelated clonal B‐cell process

Highly sensitive flow cytometry studies may incidentally identify B cell clones when used to assess plasma cell clonality in bone marrows. Clinical history, which can help differentiate related clones (low grade B cell lymphoma with plasmacytic differentiation/LBCL‐PD) from unrelated ones (plasma cell proliferative disorder (PCPD) with an unrelated B cell clone), is often unavailable in referred specimens. We sought to identify morphologic or phenotypic features that would help predict the significance of these clones in the absence of history. We included only cases with identical light chain B and plasma cell clones, as determined by 6‐color flow cytometry with additional DNA ploidy analysis, in which the relationship between clones could be established by review of medical records. There were 26 cases; 18 were related (14 were Waldenstrom macroglobulinemia) and eight were unrelated (seven multiple myeloma). Features seen exclusively in LBCL‐PD include CD19+/CD45+ clonal plasma cell phenotype (66·7%, P = 0·0022) and morphologic features such as paratrabecular bone marrow involvement, increased mast cells, and plasma cells surrounding B‐cell nodules. Aneuploidy was identified exclusively in PCPD cases (75%, P = 0·000028). We conclude that CD19+/CD45+ clonal plasma cell phenotype and aneuploidy are useful in distinguishing related clones (LBCL‐PD) from unrelated clones (PCPD).     

Immune reconstitution syndrome in patients treated for HIV and tuberculosis in Rio de Janeiro.

We made a retrospective longitudinal study from January 2000 to January 2003 to examine cases of immune reconstitution syndrome (IRS) and its incidence rate in tuberculosis (TB)-human immunodeficiency virus (HIV) co-infected patients. The incidence rate (IR) was calculated using a Poisson regression. The confidence interval (CI) that was stipulated was 95%. IRS occurred in 10/84 HIV and TB-positive patients; nine of them were on highly active anti-retroviral therapy (HAART) during a mean of 61.7 (+/- 59) days following the introduction of antiretrovirals. Lymph-node enlargement was the sole clinical manifestation. CD4 counts were <100 cells/mm(3)in 50% of the patients, at the time of TB diagnosis. All but two patients were treated with prednisone, and recovered from TB within a mean of 91 days (+/- 30 days). One relapse of TB was observed, but there were no IRS-related deaths. The incidence rate was higher (IR=11.18; CI, 1.41-88.76) in patients that had superficial lymph node enlargement at the moment of TB diagnosis (not associated with TB), extrapulmonary TB (IR=1.97; CI, 0.44-8.79), were antiretroviral naive (IR=1.85; CI, 0.48-7.16), and CD4 counts <100 cells/mm(3) (IR=1.50; CI, 0.40-5.59), although with a wide CI. IRS was frequent in our sample, occurred more frequently in HIV-naive patients with lymph-node enlargement and extrapulmonary TB. No cases of new pulmonary lesions or worsening of pulmonary infiltrates were observed.     

Oxysterols and EBI2 promote osteoclast precursor migration to bone surfaces and regulate bone mass homeostasis

Bone surfaces attract hematopoietic and nonhematopoietic cells, such as osteoclasts (OCs) and osteoblasts (OBs), and are targeted by bone metastatic cancers. However, the mechanisms guiding cells toward bone surfaces are essentially unknown. Here, we show that the Gαi protein–coupled receptor (GPCR) EBI2 is expressed in mouse monocyte/OC precursors (OCPs) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-OHC) is secreted abundantly by OBs. Using in vitro time-lapse microscopy and intravital two-photon microscopy, we show that EBI2 enhances the development of large OCs by promoting OCP motility, thus facilitating cell–cell interactions and fusion in vitro and in vivo. EBI2 is also necessary and sufficient for guiding OCPs toward bone surfaces. Interestingly, OCPs also secrete 7α,25-OHC, which promotes autocrine EBI2 signaling and reduces OCP migration toward bone surfaces in vivo. Defective EBI2 signaling led to increased bone mass in male mice and protected female mice from age- and estrogen deficiency–induced osteoporosis. This study identifies a novel pathway involved in OCP homing to the bone surface that may have significant therapeutic potential.Osteoclasts (OCs) are multinucleated cells that regulate skeletal development and integrity by actively resorbing excess or damaged bone produced by osteoblasts (OBs) and osteocytes. OBs and osteocytes differentiate from rare mesenchymal stem cells that reside in BM parenchyma (Méndez-Ferrer et al., 2010). In contrast, OCs differentiate from BM-resident and circulatory monocytic precursors that come into close contact with bone surfaces where the essential cytokines ligand for receptor activator of nuclear factor kappa binding (RANKL, encoded by Tnfsf11) and M-CSF (encoded by Csf1) are locally produced by OBs and osteocytes (Teitelbaum, 2000; Nakashima et al., 2011). Cellular interactions between OCs and OBs regulate the activity of both cell types, such that resorbed bone is accurately replaced by newly formed bone. Although the understanding of the molecular signals required for OC differentiation has increased significantly over the past several decades, very little is understood about the mechanisms controlling the migration and positioning of OC precursors (OCPs) near, or in contact with, the bone surface.Monocytes and OCPs are dynamic within BM parenchyma (Ishii et al., 2009). OCPs recirculate between BM and peripheral organs via the action of sphingosine 1-phosphate receptors (S1PRs), which attract multiple hematopoietic cell subsets from BM parenchyma into blood circulation (Walzer et al., 2007; Ishii et al., 2009, 2010; Pereira et al., 2010b). In contrast, the signals promoting cell movement toward bone surfaces remain unknown. However, systemic RANKL administration has been shown to increase OCP homing back to BM and to promote local OC differentiation (Kotani et al., 2013). These studies suggest that OCP movement in and out of BM tissue is highly regulated and that balanced responsiveness to various chemoattractants regulates OCP movement and differentiation. Consistent with this hypothesis, bone resorption produces a milieu that contains potent chemoattractants for monocytes/OCPs (Mundy et al., 1978).Here, we investigated the role played by EBI2, a Gαi protein–coupled receptor (GPCR) involved in dendritic cell and B lymphocyte migration in secondary lymphoid organs (Gatto et al., 2009, 2013; Pereira et al., 2009b; Hannedouche et al., 2011; Kelly et al., 2011; Liu et al., 2011; Yi and Cyster, 2013), in controlling monocyte and OCP movement and positioning within BM. We show that EBI2 is highly expressed in OCPs and mature OCs and promotes OCP motility in vitro and in vivo. Furthermore, we show that OCPs deficient in EBI2 migrate poorly toward bone surfaces, which reduces OC differentiation. In contrast, OCPs that overexpressed EBI2 preferentially localized at the bone surface and fused with preexisting OCs more efficiently than control OCPs. OBs expressed the enzymes CH25H and CYP7B1 that are required for the synthesis of the EBI2-ligand 7α,25-dihydroxycholesterol (7α,25-OHC) and secreted EBI2 ligands in vitro. Interestingly, OCPs also secreted 7α,25-OHC, which results in autocrine EBI2 signaling and tempers EBI2-mediated migration toward bone surfaces. Finally, EBI2 signaling–deficient mice exhibit increased bone mass at young and old ages, and EBI2 signaling deficiency significantly protects female mice from osteoporosis induced by estrogen deficiency.     

Impact of the COVID-19 Pandemic on Health-Related Concerns,Quality of Life and Psychological Adjustment in Young Adults with Congenital Heart Disease

Background: The risk for a severe disease course in case of infection with SARS-CoV-2 in young adults with congenital heart disease is largely unknown, potentially leading to uncertainty and anxiety among affected patients. This study aims to investigate health-related concerns, health-related quality of life and psychological adjustment in patients with congenital heart disease compared to healthy peers during the COVID-19 pandemic. Methods: One-hundred patients with congenital heart disease and 50 controls (M = 29.7, SD = 3.8 years) were recruited. They completed an online survey including the assessment of health-related concerns regarding COVID-19, the 12-item Short Form Health Survey and the Brief Symptom Inventory. Results: Patients considered COVID-19 to be a more serious issue (Generalized odds ratio [GenOR] = 1.67, p = 0.04), were more concerned about becoming infected (GenOR = 2.93, p < 0.001) and expressed more fear about leaving their homes (GenOR = 1.81, p = 0.004) while general anxiety symptoms were not different between groups (p = 0.23). Patients relied more on family and friends for support (30% vs. 2% in controls, p < 0.001) and reported better compliance with protective measures (p = 0.03). Mental health-related quality of life and psychological adjustment were not different between groups (p = 0.17 and p = 0.68, respectively). Physical health-related quality of life was lower in patients compared to controls (p = 0.03). Conclusions: Young adults with congenital heart disease in Switzerland are more concerned about their health during the COVID-19 pandemic compared to healthy peers. These concerns, however, do not translate into generally impaired mental wellbeing. The impact of the easing of lockdown measures on long-term anxiety levels and quality of life requires further study