Heterozygous mutations in the PALB2 hereditary breast cancer predisposition gene impact on the three‐dimensional nuclear organization of patient‐derived cell lines

PALB2/FANCN is a BRCA1‐ and BRCA2‐interacting Fanconi Anemia (FA) protein crucial for key BRCA2 genome caretaker functions. Heterozygous germline mutations in PALB2 predispose to breast cancer and biallelic mutations cause FA. FA proteins play a critical role in the telomere maintenance pathway, with telomeric shortening observed in FA cells. Less is known about telomere maintenance in the heterozygous state. Here, we investigate the roles of PALB2 heterozygous mutations in genomic instability, an important carcinogenesis precursor. Patient‐derived lymphoblastoid (LCL) and fibroblast (FCL) cell lines with monoallelic truncating PALB2 mutations were investigated using a combination of molecular imaging techniques including centromeric FISH, telomeric Q‐FISH and spectral karyotyping (SKY). Mitomycin C and Cisplatin sensitivity was assayed via cellular metabolism of WST‐1. The PALB2 c.229delT FCL showed increases in telomere counts associated with increased mean intensity compared with two wild‐type FCLs generated from first‐degree relatives (P =1.04E‐10 and P =9.68E‐15) and it showed evidence of chromosomal rearrangements. Significant differences in centromere distribution were observed in one of three PALB2 heterozygous FCLs analyzed when compared with PALB2 wild‐type, BRCA1 and BRCA2 heterozygous FCLs. No significant consistently increased sensitivity to Mitomycin C or Cisplatin was observed in LCLs. Our results are suggestive of an altered centromere distribution profile and a telomere instability phenotype. Together, these may indicate critical nuclear organization defects associated with the predisposition to transformation and early stage development of PALB2‐related cancers. © 2013 Wiley Periodicals, Inc.     

Proteomic analysis of mantle-cell lymphoma by protein microarray

Mantle-cell lymphoma (MCL) is a unique subtype of B-cell non-Hodgkin lymphoma (NHL) that behaves aggressively and remains incurable. In order to understand the pathogenesis of MCL and design new therapies, it is important to accurately analyze molecular changes in pathways dysregulated in MCL. We used antibody microarrays to compare patterns of protein expression between CD19(+) purified B lymphocytes from normal tonsil and 7 cases of histologically confirmed MCL. Protein overexpression was defined as a higher than 1.3-fold or 2-fold increase in at least 67% of tumor samples compared with normal B-cell control. Of the polypeptides, 77 were overexpressed using the higher than 1.3-fold cutoff, and 13 were overexpressed using the 2-fold cutoff. These included cell cycle regulators (regulator of chromosome condensation 1 [RCC1], murine double minute 2 [MDM2]), a kinase (citron Rho-interacting kinase [CRIK]), chaperone proteins (heat shock 90-kDa protein [Hsp90], Hsp10), and phosphatase regulators (A-kinase anchor protein 1 [AKAP149], protein phosphatase 5 [PP5], and inhibitor 2). The elevated expression of some of these polypeptides was confirmed by immunoblotting and immunohistochemistry, whereas elevated expression of others could not be confirmed, illustrating the importance of confirmatory studies. This study describes a novel technique that identifies proteins dysregulated in MCL.     

Evaluation of the Patient-Specific Functional Scale in hand fractures and dislocations

Background

Generic upper extremity disability questionnaires utilize standardized items. The Patient-Specific Functional Scale (PSFS) allows the patient to identify specific self-reported items. This study evaluated the validity of the PSFS to assess outcome in patients with hand fractures or dislocations.

Methods

Adults with hand fractures or dislocations, who completed hand therapy between January 2012 and January 2013, were eligible for inclusion. At the initial and final assessment, each patient was asked to complete the PSFS. Each patient identified three items that were difficult or they were unable to perform, and the degree of difficulty was ranked from 0 to 10 (able to perform at pre-injury level). We excluded patients with an incomplete PSFS. Statistical analyses evaluated the relationships between the PSFS and the independent variables.

Results

There were 63 patients (37 men, 26 women); 21 of the 63 patients underwent surgery for fracture fixation. The mean duration of hand therapy treatment was 2.2 ± 1.4 months. The mean PSFS scores were as follows: initial 3.2 ± 2.2; final 8.1 ± 2.2. There was a significant improvement in PSFS scores from initial to final assessment (p < 0.001) and a moderate correlation (r = 0.3, p = 0.02). There was no statistical difference in PSFS scores between men and women or surgery and no surgery.

Conclusions

In these patients with hand fractures or dislocations, the PSFS indicated significant improvement in function. Using items identified by the patient, the PSFS provides a valuable perspective of outcome and may be used in conjunction with generic disease-specific questionnaire for assessment of the upper extremity.     

Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4

Heparin-induced thrombocytopenia (HIT) is an important complication of heparin therapy. Although there is general agreement that platelet activation in vitro by the HIT IgG is mediated by the platelet Fc receptor, the interaction among the antibody, heparin, and platelet membrane components is uncertain and debated. In this report, we describe studies designed to address these interactions. We found, as others have noted, that a variety of other sulfated polysaccharides could substitute for heparin in the reaction. Using polysaccharides selected for both size and charge, we found that reactivity depended on two independent factors: a certain minimum degree of sulfation per saccharide unit and a certain minimum size. Hence, highly sulfated but small (< 1,000 daltons) polysaccharides were not reactive nor were large but poorly sulfated polysaccharides. The ability of HIT IgG to recognize heparin by itself was tested by Ouchterlony gel diffusion, ammonium sulfate and polyethylene glycol precipitation, and equilibrium dialysis. No technique demonstrated reactivity. However, when platelet releasate was added to heparin and HIT IgG, a 50-fold increase in binding of radio-labeled heparin to HIT IgG was observed. The releasate was then depleted of proteins capable of binding to heparin by immunoaffinity chromatography. Only platelet factor 4-immunodepleted releasate lost its reactivity with HIT IgG and heparin. Finally, to determine whether the reaction occurred on the surface of platelets or in the fluid phase, washed platelets were incubated with HIT IgG or heparin and after a wash step, heparin or HIT IgG was added, respectively. Reactivity was only noted when platelets were preincubated with heparin. Consistent with these observations was the demonstration of the presence of PF4 on platelets using flow cytometry. These studies indicate that heparin and other large, highly sulfated polysaccharides bind to PF4 to form a reactive antigen on the platelet surface. HIT IgG then binds to this complex with activation of platelets through the platelet Fc receptors.     

Epidemiology of Sexual Health in the Virtual Environment: A Multinational Online Survey of Spanish- and Portuguese-Speaking Men who use an Internet Sexual Networking Site

There is limited data on the sexual health of users of sexual networking websites for men who have sex with men (MSM) in Latin America. Members of a MSM-targeted social/sexual networking website in Latin America, Spain, and Portugal participated in an online sexual health survey. Among 36,063 respondents, nearly 90 % reported having anal or vaginal intercourse in the past 3 months. Among sexually active men, 53.2 % used condoms inconsistently. In the past year, 54 % of respondents reported undergoing sexually transmitted infections (STI) testing and 67 % reported testing yearly or more often for HIV. Self-reported HIV prevalence was 9.1 %. Differences were seen by geographic region. Unprotected intercourse with partners of different or unknown HIV status was associated with recent STI diagnosis (OR = 1.83, t = 13.15, d.f. = 21, p < .001) and HIV diagnosis (OR = 2.20, t = 14.00, d.f. = 21, p < .001). Online surveys are a promising tool for HIV/STI surveillance and prevention internationally to reach an important subset of MSM.     

Mutation in Osteoactivin Decreases Bone Formation in Vivo and Osteoblast Differentiation in Vitro

We have previously identified osteoactivin (OA), encoded by Gpnmb, as an osteogenic factor that stimulates osteoblast differentiation in vitro. To elucidate the importance of OA in osteogenesis, we characterized the skeletal phenotype of a mouse model, DBA/2J (D2J) with a loss-of-function mutation in Gpnmb. Microtomography of D2J mice showed decreased trabecular mass, compared to that in wild-type mice [DBA/2J-Gpnmb⁺/SjJ (D2J/Gpnmb⁺)]. Serum analysis showed decreases in OA and the bone-formation markers alkaline phosphatase and osteocalcin in D2J mice. Although D2J mice showed decreased osteoid and mineralization surfaces, their osteoblasts were increased in number, compared to D2J/Gpnmb⁺ mice. We then examined the ability of D2J osteoblasts to differentiate in culture, where their differentiation and function were decreased, as evidenced by low alkaline phosphatase activity and matrix mineralization. Quantitative RT-PCR analyses confirmed the decreased expression of differentiation markers in D2J osteoblasts. In vitro, D2J osteoblasts proliferated and survived significantly less, compared to D2J/Gpnmb⁺ osteoblasts. Next, we investigated whether mutant OA protein induces endoplasmic reticulum stress in D2J osteoblasts. Neither endoplasmic reticulum stress markers nor endoplasmic reticulum ultrastructure were altered in D2J osteoblasts. Finally, we assessed underlying mechanisms that might alter proliferation of D2J osteoblasts. Interestingly, TGF-β receptors and Smad-2/3 phosphorylation were up-regulated in D2J osteoblasts, suggesting that OA contributes to TGF-β signaling. These data confirm the anabolic role of OA in postnatal bone formation.Osteoporosis is a growing public health problem, in part because of the increasing numbers of people living beyond the age of 65 years.¹ It is characterized by low bone mass due to increased bone resorption by osteoclasts and decreased bone formation by osteoblasts, with significant deterioration in the bone microarchitecture leading to high bone fragility and increased fracture risk.^1,2 The net effect of osteoporosis is low bone mass.¹ There is an increasing demand for identifying novel bone anabolic factors with potential therapeutic benefits in treating generalized bone loss, such as osteoporosis and/or major skeletal fracture.Osteoactivin is a novel glycoprotein first identified in natural mutant osteopetrotic rats.³ The same protein has been identified and named separately in several other species: as dendritic cell heparan sulfate proteoglycan integrin dependent ligand (DCHIL) in mouse dendritic cells,⁴ as transmembrane glycoprotein NMB (GPNMB) in human melanoma cell lines and melanocytes,⁵ and as hematopoietic growth factor inducible neurokinin (HGFIN) in human tumor cells.⁶ The current recommended name for the protein encoded by Gpnmb in mouse is transmembrane glycoprotein NMB (http://www.ncbi.nlm.nih.gov/protein/Q99P91.2); here, we continue to use osteoactivin (OA) for the protein and Gpnmb for the gene. OA is a type I transmembrane protein that consists of multiple domains, including an extracellular domain, transmembrane domain, and protein sorting signal sequence.⁷ Within the C-terminal domain, OA has an RGD motif, predicting an integrin attachment site.^3,7–9Our research group initially reported on the novel role of OA in osteoblast differentiation and function.^7–10 We demonstrated that OA expression has a temporal pattern during osteoblast differentiation, being highest during matrix maturation and culture mineralization in vitro.^7–11 Using loss-of–function and gain-of–function approaches in osteoblasts, we reported that OA overexpression increases osteoblast differentiation and function and that OA down-regulation decreases nodule formation, alkaline phosphatase (ALP) activity, osteocalcin (OC) production, and matrix mineralization in vitro.⁷ We also reported on the positive role of OA in mesenchymal stem cell (MSCs) differentiation into osteoblasts in vitro.¹² In another study, we showed that recombinant OA protein induces higher osteogenic potential of fetal-derived MSCs, compared with bone marrow–derived MSCs¹³ and its osteogenic effects in the mouse C3H10T1/2 MSC cell line were similar to those of recombinant BMP-2.¹² We also localized OA protein as associated predominately with osteoblasts lining trabecular bones in vivo,¹¹ and showed that local injection of recombinant OA increased bone mass in a rat model.¹⁴ Moreover, in a fracture repair model OA expression increased over time, reaching a maximum 2 weeks after fracture.¹¹ In a parallel study, recombinant OA supported bone regeneration and formation in a rat critical-size calvarial defect model.¹⁵ Others have shown that OA is highly expressed by osteoclasts in vitro, suggesting that it may regulate osteoclast formation and activity.¹⁶There is urgent need for an animal model to fully examine the role of OA in osteogenesis. Interestingly, a natural mutation of the Gpnmb gene has been identified in the DBA/2J (D2J) mouse strain.¹⁷ These mice exhibit high-frequency hearing loss, which begins at the time of weaning and becomes severe by 2 to 3 months of age.^18,19 Aged D2J mice also develop progressive eye abnormalities that closely mimic human hereditary glaucoma. The onset of disease symptoms falls roughly between 3 and 4 months of age, and disease becomes severe by 6 months of age.^5,20 D2J mice are homozygous for a nonsense mutation in the Gpnmb gene sequence that induces an early stop codon, generating a truncated protein sequence of 150 amino acids (aa) instead of the full-length 562-aa OA protein.⁵ The control for the D2J mouse is the wild-type DBA/2J-Gpnmb⁺/SjJ mouse (D2J/Gpnmb⁺), homozygous for the wild-type Gpnmb gene.²¹ These Gpnmb wild-type mice do not develop glaucoma, as D2J mice do, although they exhibit mild iris stromal atrophy.²¹In the present study, we used Gpnmb mutant (D2J) and Gpnmb wild-type (D2J/Gpnmb⁺) mice to gain insight into the role of OA in osteogenesis and in osteoblast differentiation and function. Here, we report that loss-of–function mutation of Gpnmb suppresses bone formation by directly affecting osteoblast proliferation and survival, leading to a decreased number of differentiated osteoblasts with suppressed activity in bone mineralization. Thus, our data point to OA as a novel and positive regulator of postnatal bone formation.     

Macrophage activation and skeletal muscle healing following traumatic injury

Following injury to different tissues, macrophages can contribute to both regenerative and fibrotic healing. These seemingly contradictory roles of macrophages may be related to the markedly different phenotypes that macrophages can assume upon exposure to different stimuli. We hypothesized that fibrotic healing after traumatic muscle injury would be dominated by a pro‐fibrotic M2a macrophage phenotype, with M1 activation limited to the very early stages of repair. We found that macrophages accumulated in lacerated mouse muscle for at least 21 days, accompanied by limited myofibre regeneration and persistent collagen deposition. However, muscle macrophages did not exhibit either of the canonical M1 or M2a phenotypes, but instead up‐regulated both M1‐ and M2a‐associated genes early after injury, followed by down‐regulation of most markers examined. Particularly, IL‐10 mRNA and protein were markedly elevated in macrophages from 3‐day injured muscle. Additionally, though flow cytometry identified distinct subpopulations of macrophages based on high or low expression of TNFα, these subpopulations did not clearly correspond to M1 or M2a phenotypes. Importantly, cell therapy with exogenous M1 macrophages but not non‐activated macrophages reduced fibrosis and enhanced muscle fibre regeneration in lacerated muscles. These data indicate that manipulation of macrophage function has potential to improve healing following traumatic injury. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.