Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression, and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non‐24‐hour sleep‐wake disorders, and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT₁ and MT₂, belonging to the G protein‐coupled receptor (GPCR) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved to be difficult to obtain. Here, we describe the development of the first monoclonal antibodies (mABs) for mouse MT₁ and MT₂. Purified antibodies were extensively characterized for specific reactivity with mouse, rat, and human MT₁ and MT₂ by Western blot, immunoprecipitation, immunofluorescence, and proximity ligation assay. Several mABs were specific for either mouse MT₁ or MT₂. None of the mABs cross‐reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR‐KO mice as control. MT₂ expression was not detected in mouse insulinoma MIN6 cells or pancreatic beta‐cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies.     

Increased risk of osteoporosis and fracture in women with systemic sclerosis: A comparative study with rheumatoid arthritis

Objective

To investigate whether women with systemic sclerosis (SSc) have an increased risk of osteoporosis (OP) and related fractures compared to a high‐risk population with rheumatoid arthritis (RA) and also healthy controls, and to determine putative specific OP and fracture risk factors.

Methods

We performed a cross‐sectional study with successive inclusion of age‐matched healthy women and women with SSc and RA. Risk factors for OP and fracture were collected for all patients. Bone mineral density (BMD) was systematically measured at the lumbar spine and total hip region with dual x‐ray absorptiometry.

Results

We included 71 women with SSc, 139 women with RA, and 227 healthy women. The prevalence of OP and fracture was similar in SSc and RA, and was for both diseases higher than in healthy controls (OP: 30% in SSc, 32% in RA, and 11% in controls; fracture: 35% in SSc, 33% in RA, and 10% in controls). Multivariate analysis identified age as a risk factor of OP in SSc. Age and low 25‐hydroxyvitamin D (25[OH]D) levels were recognized as risk factors of fracture in SSc. In comparison, age and corticosteroid treatment were associated with OP in RA. Multivariate analysis confirmed age, OP, and low 25(OH)D levels as independent risk factors of fractures in RA.

Conclusion

The prevalence of OP and fracture in SSc was increased compared to healthy women and reached the high prevalence associated with RA. Age and vitamin D deficiency were identified as risk factors of fracture in SSc. Therefore, increasing the awareness and performance of BMD measurements together with the vitamin D supply in patients with SSc is warranted.     

Triaging in pulmonary embolism

Risk stratification of patients with pulmonary embolism represents an important step and may help to guide initial therapeutic management. Pulmonary embolism can be stratified into several groups, with different risk of early death or complications based on the presence of several risk factors. High-risk pulmonary embolism is defined by shock or peripheral signs of hypoperfusion. It is a life-threatening emergency with high short-term mortality (>25%) requiring specific therapeutic strategy with inotropic agents and fibrinolysis. In normotensive patients with pulmonary embolism, the presence of right ventricular dysfunction assessed by echocardiography or myocardial injury based on elevated levels of biomarkers, is associated with an intermediate risk of early death. These patients require close monitoring, and the role of thrombolytic treatment is currently assessed in a large trial. Lastly, patients with normotensive pulmonary embolism and without right ventricular dysfunction or myocardial injury have a low risk of death and complications. These patients may be candidates for home treatment. Several scores combining these risk factors have been described.     

Microsatellite instability mutator phenotype in hepatocellular carcinoma in non-alcoholic and non-virally infected normal livers

Microsatellite instability (MSI) seems to be a rare event in hepatocarcinogenesis and might actually be associated with the progression of hepatocellular carcinoma (HCC) in which the liver is often the site of chronic hepatitis or cirrhosis. The aim of this work was to define the MSI phenotype in HCC affecting exclusively normal livers to avoid slippage errors due to cirrhosis. One hundred and sixty-four patients with HCC affecting non-cirrhotic livers were operated on in our hospital between 1984 and 2001. We analyzed 37 patients selected for low alcohol consumption and the absence of HBV or HCV infection. All the livers were histologically normal. MSI was analyzed according to the criteria defined during the conference consensus workshop for colorectal cancer. High MSI (MSI-H > 30%) was found in 6 (16%) and low MSI (MSI-L < 30%) in 10 (27%) of the 37 HCCs. None of the 10 microsatellite markers tested were altered in the remaining 21 tumors (57%). Immunohistochemistry showed that normal amounts of hMLH1 and hMSH2 were present both in MSI-H and in MSI-L HCCs. MSI-H was significantly associated with more aggressive histological tumor features and a shorter median delay before recurrence. Thus, we have found a small subgroup of HCC tumors which can be considered as a new clinical/histological entity.     

Long-term results and competing risk analysis of the H89 trial in patients with advanced-stage Hodgkin lymphoma: a study by the Groupe d'Etude des Lymphomes de l'Adulte (GELA)

From 1989 to 1996, 533 eligible patients with stage IIIB/IV Hodgkin lymphoma (HL) were randomly assigned to receive 6 cycles of hybrid MOPP/ABV (mechlorethamine, vincristine, procarbazine, prednisone/Adriamycin [doxorubicin], bleomycin, vinblastine; n = 266) or ABVPP (doxorubicin, bleomycin, vinblastine, procarbazine, prednisone; n = 267). Patients in complete remission (CR) or partial response of at least 75% after 6 cycles received 2 cycles of consolidation chemotherapy (CT) (n = 208) or subtotal nodal irradiation (RT) (n = 210). A better survival probability was observed after ABVPP alone: the 10-year overall survival (OS) estimates were 90% for ABVPP x 8, 78% for MOPP/ABV x 8, 82% for MOPP/ABV with RT, and 77% for ABVPP x 6 with RT (P = .03); and the 10-year disease-free survival (DFS) estimates were 70%, 76%, 79%, and 76%, respectively (P = .09). The 10-year DFS estimates for patients treated with consolidation CT or RT were 73% and 78% (P = .07), and OS estimates were 84% and 79%, respectively (P = .29). These results showed that RT was not superior to consolidation CT after a doxorubicin-induced CR in patients with advanced HL. An analysis of competing risks identified age more than 45 years as a significant risk factor for death, relapse, and second cancers. Prospective evaluation of late adverse events may improve the management of patients with HL.     

Double-read of skeletal surveys in suspected non-accidental trauma: what we learned

Background

Missing a fracture in a child on skeletal surveys for suspected non-accidental trauma can have devastating results. Double-read has the potential to improve fracture detection. However the yield of double-read is unknown.

Objective

To determine the advantage of double-read versus single-read of radiographic skeletal surveys for suspected non-accidental trauma.

Materials and methods

The study was performed in two phases. In the first phase (April 2013 to September 2013), double-read was performed for all skeletal surveys obtained during weekday working hours. Because we had no new double-read findings in studies initially read as negative, we conducted a second phase (January 2014 to March 2014). In the second phase we limited double-reads to skeletal surveys found positive on the first read. At the end of this period, we retrospectively performed double-read for all initially negative skeletal surveys. We excluded follow-up skeletal surveys. The difference in discrepancy (new fracture or false diagnosis of a fracture) ratio between negative and positive skeletal surveys was evaluated using the Fisher exact test, and change in discrepancy ratio between the first and second study phases was evaluated using the stratified Cochran-Mantel-Haenszel test.

Results

Overall in the two phases, 178 skeletal surveys were performed in 178 children (67 girls) with mean age of 9 months (range 3 days to 3.7 years). Double-read found 16 discrepancies in 8/178 (4.5%) skeletal surveys. Seven of these studies showed additional fractures (n=15). In one study, an initial read of a skull fracture was read as a variant on the second read. There was a significant (P=0.01) difference between rate of disagreement in negative skeletal surveys (1/104, 1.0%) and positive skeletal surveys (7/74, 9.5%). No significant change in disagreement rate was demonstrated between the two phases of the study (P=0.59).

Conclusion

Double-read of skeletal survey for suspected non-accidental trauma found false-negative fractures in a few cases and rarely found false-positive diagnosis of a fracture. Double-read uncommonly found discrepancies in an initially normal skeletal survey. Limiting double-read to initially positive studies improves the yield of the double-read.