Cryptococcosis diagnosis has been recently improved by the use of rapid cryptococcal antigen testing with lateral flow assays, which have proved sensitive and specific. Using “test and treat” screening strategies for cryptococcal disease with these tests has been showed effective in reducing cryptococcal meningitis (CM) in HIV-infected patients. Recommended induction, consolidation, and maintenance therapeutic strategy for CM is widely unavailable and/or expensive in low and middle-income settings. New therapeutic strategies, mostly using reduced duration, have recently shown acceptable outcome or are currently tested. Diagnostic and therapeutic guidelines for cryptococcal disease in limited resources countries are undergoing a paradigmatic shift. The diagnosis of cryptococcal meningitis (CM) and more broadly, disseminated cryptococcosis, is easily made by direct examination of a pelleted biological fluid using India ink with the typical observation of rounded cells measuring about 3-10 μm in diameter surrounded a thick capsule represented as a clear halo. Sensitivity of this technique is around 86%  but can However, cryptococcosis is a sub-acute infection. Consequently, CrAg can be detected in blood many weeks before the onset of symptoms . Serum CAs such, systematic pre-ART CrAg screening and preemptive fluconazole is recommended for those presenting with < 100 CD4 cell counts . Though CrAg detection and diagnosis of cryptococcal disease - Commercially available CrAg detection tests are highly sensitive and specific in serum and CSF in predicting the presence of meningitis in HIV positive patients presenting with evocative symptoms. CrAg detection in serum and CSF with symptoms suggestive of CM strongly correlates with culture- and/or India ink-confirmed CM. CSF CrAg is a rapid and reliable diagnostic test for confirmed HIV-associated CM  . CrAg-positive patients who were not offered fluconazole pre-emptive therapy are at higher risk of CM and death , is measured as the rate of yeast clearance per millilitre of CSF per day based on CFU counting. In a combined cohort of 262 patients, a slow rate of cryptococcal clearance was shown to be independently associated with increased mortality at two weeks and at 10 weeks . Though,Thereafter, we summarise the main studies and clinical trials which have helped to improve cryptococcosis outcome and survival in the last decades.Amphotericin B with flucytosine - The use of flucytosine combined with amphotericin B for CM therapy was already observed in a study in 1979, before the HIV pandemic, where authors concluded that the combination therapy allowed more rapid sterilisation of the cerebrospinal fluid (p < 0.001) and less nephrotoxicity (p < 0.05) than did amphotericin B alone, although results were obtained at different dosages than those currently recommended  was shown to be significantly faster in the arm in which patients were treated with the combination of amphotericin B plus flucytosine than in any of the three other arms , in 64 HIV-infected patients with CM .This strategy was then also shown to be superior in the French prospective CryptoA/D study in HIV-positive or HIV-negative patients with cryptococcal infection with a mycological failure at week two of 26% in the amphotericin B and flucytosine group compared to a treatment failure of 56% with any other treatments (p < 0.001) .Moreover, a three-arm, open-label, randomised trial including about 300 HIV-infected patients enrolled, demonstrated a survival benefit with this combination compared to amphotericin alone, with a reduction of about 40% in the relative risk of death at 10 weeks with addition of flucytosine .Finally, the recently completed “Advancing Cryptococcal Meningitis Treatment for Africa” (ACTA) trial, in which 721 HIV-infected patients with first episode of CM were randomised over nine sites in four different countries in Sub-Saharan Africa, definitively evidenced that the combination of amphotericin B and flucytosine was significantly superior to Amphotericin B and fluconazole, leading to a substantial mortality reduction  .Amphotericin B with fluconazole - When flucytosine is unavailable, the combination of amphotericin B with fluconazole is still recommended  in induction therapy associated with amphotericin B was tested in a randomised 4-arms trial and found to have similar EFA, and could therefore be a theoretical option for therapy, despite its higher cost and potential interactions with other drugs including rifampicin (fampicin . As voriShort courses therapy - The amphotericin B and flucytosine combination regimen remains unavailable in most parts of the world harbouring the highest burden of disease (Africa and Asia), furthermore several days of amphotericin B deoxycholate is well known to cause important adverse events, such as anaemia, renal impairment, hypokalaemia, hypomagnesemia, or phlebitis, and its administration requires inpatient hospitalisation for intravenous administration and electrolytes monitoring ) . This prFluconazole and flucytosine - Before the highly active antiretroviral therapy era, an open-label, single-arm, prospective trial studied the fluconazole (400 mg daily) and flucytosine (150 mg/kg daily) combination therapy for a 10-week antifungal therapy in 32 patients with AIDS, and found a clinical survival at 10 weeks which appeared to be better than previously reported with amphotericin B alone or fluconazole alone  and flucytosine (100 mg/kg daily) to high dose fluconazole alone for the 2-week induction therapy, in 41 HIV-infected patients with CM in Malawi, and found better EFA and survival for the combination therapy arm .Finally, the ACTA trial including 721 randomised HIV-infected patients also showed that a combination oral therapy with high dose fluconazole (1200 mg/day) and flucytosine (100 mg/kg/day) for the 2-week induction therapy was non-inferior to the standard 2-week courses of amphotericin B based therapy with flucytosine or fluconazole . That maBoth 2010 IDSA and 2011 WHO guidelines recommend high-dose fluconazole monotherapy (1200 mg/ day) for 10-12 weeks if amphotericin and flucytosine are not available .In a cohort study carried out in Malawi using high dose fluconazole (1200 mg) as induction therapy , mortaliA recently published CrAg screening study in Ethiopia in HIV-infected patients, highlighted that the use of high dose fluconazole monotherapy for induction phase is inadequate, leading to a 68% mortality rate at three months, in patients with CM .At the beginning of highly active antiretroviral therapy era, in 1997, a randomised trial comparing liposomal amphotericin B (L-AmB) and conventional deoxycholate amphotericin B in 28 HIV-infected patients with CM, observed that a 3-week course of 4 mg/kg L-AmB had a significantly earlier CSF culture conversion than a 3-week course standard amphotericin B, with similar clinical outcome and that L-AmB had significantly less renal adverse events .In a larger multi-centre trial, in which 267 patients were randomised, liposomal amphotericin B - therapy was similar in term of efficacy and overall mortality at 10 weeks, compared to conventional amphotericin B deoxycholate for HIV-associated CM. L-AmB at a dosage of 3 mg/kg/day was associated with significantly fewer adverse effects than at a dosage of 6 mg/kg/day, and compared to conventional amphotericin B .More recently, 80 participants were enrolled in the AMBITION preliminary phase II trial comparing different short courses of L-AmB with high dose fluconazole. The single dose arm was non-inferior to the standard 14-days arm in term of early fungicidal activity, compared to two-doses or three-doses arms .Based on these results, the AMBITION phase III (ISRCTN 72509687) ongoing trial is currently taking place to evaluate whether a single high dose (10 mg/kg) of LAmB given with high dose fluconazole and flucytosine is as effective as the newly recommended  one-week daily-dosed amphotericin B deoxycholate based induction therapy with flucytosine as an induction therapy . This trSteroids - Adjuvant glucocorticoid therapy is successfully used in patients with non-fungal meningitis such as bacterial or tuberculous meningitis, and reduces mortality in some sub-groups. In CM IDSA guidelines  for safety reasons . All patInterferon-gamma - In a prospective trial, with 90 HIV-infected patients randomised in three different arms , an increased rate of clearance with two doses of adjunctive INF-γ than with standard therapy was shown, but there was no significant difference in mortality between groups , frequently used as an anti-depressant, has a real in vitro and in vivo fungicidal activity against Cryptococcus neoformans, as its action has a synergistic effect with fluconazole in reducing the fungal burden in brain, kidney, and spleen ” phase III randomised placebo-controlled clinical trial had been evaluating if sertraline dosed initially at 400 mg/day for two weeks, followed by 200 mg/day for an additional 10 weeks prior to tapering, could have an 18-week survival benefit, compared with placebo when receiving standard available induction therapy of amphotericin B deoxycholate and fluconazole 800 mg/day. The trial was stopped after enrolling 460 of the 550 patients planned for futility, showing a 18-week mortality of 52% in sertraline group and 46% in placebo group (p = 0.15) .Tamoxifen - Tamoxifen, the oestrogen receptor antagonist drug usually used for breast cancer, has been shown to be fungicidal and synergistic with fluconazole and amphotericin B in vitro and in vivo in a mouse model of disseminated cryptococcosis  will enrol 50 patients in Vietnam, comparing EFA according to the strategy between (amphotericin B plus fluconazole ± tamoxifen during the first two weeks).Elevated intracranial pressure (CSF pressure > 25 cm H2O) is a usual complication of CM, secondary to a failure of CSF resorption because of obstruction by crypto-coccal polysaccharide capsule and yeasts.In several large randomised trials  of amphoAnother randomised trial comparing acetazolamide or placebo, with amphotericin B alone was prematurely stopped after recruitment of 24 patients in Thailand with CM, presenting with headache and an elevated opening cerebrospinal fluid pressure ≥ 20 cm H20, after having observed that patients who received acetazolamide developed significantly lower venous bicarbonate levels and higher chloride levels and had more frequent serious adverse events than patients who received placebo .In the COAT trial, the effect of therapeutic lumbar puncture (LP) was evaluated in HIV-infected patients treated for CM, and investigators found that therapeutic LPs were associated with a 69% relative improvement in survival, regardless of initial intracranial pressure .Antiretroviral therapy (ART) is the corner stone of treatment in HIV-infected patients with advanced AIDS disease. In patients with CM, it is now recommended delaying ART for four-six weeks after starting antifungal therapy . This deIn solid organ transplant recipients, cryptococcal disease has different presentation, with less disseminated disease, and more lung-limited presentation . This coCryptococcal disease is a death-related disease in immunosuppressed patients, especially in advanced AIDS disease patients. Over the past 15 years, many studies and clinical trials have led to improve prevention, diagnosis, therapy and outcome of patients with cryptococcal meningitis. Efforts are now needed to implement those strategies in middle and low-income settings using affordable and efficient diagnostic strategies and drug regimen. These new strategies are being evaluated and will be implemented progressively to improve patient management.


Non–ST-segment elevation myocardial infarction (NSTEMI) comprises the majority of MI worldwide, yet mortality remains high. Management of NSTEMI is relatively delayed and heterogeneous compared with the “time is muscle” approach to ST-segment elevation MI, though it is unknown to what extent comorbid conditions drive NSTEMI mortality.We sought to quantify mortality due to MI versus comorbid conditions in patients with NSTEMI.Participants of the ARIC (Atherosclerosis Risk in Communities) study cohort ages 45 to 64 years, who developed incident NSTEMI were identified and incidence-density matched to participants who did not experience an MI by age group, sex, race, and study community. We estimated hazard ratios for all-cause mortality, comparing those who developed NSTEMI to those who did not experience an MI.ARIC participants with incident NSTEMI were more likely at baseline to be smokers, have diabetes and renal dysfunction, and take blood pressure or cholesterol-lowering medications than were participants who did not have an MI. Over one-half of participants experiencing NSTEMI died over a median follow-up of 8.4 years; incident NSTEMI was associated with 30% higher risk of mortality after adjusting for comorbid conditions .NSTEMI confers a significantly higher mortality hazard beyond what can be attributed to comorbid conditions. More consistent and effective strategies are needed to reduce mortality in NSTEMI amid comorbid conditions. Ischemic heart disease, often manifest as myocardial infarction (MI), is the leading cause of disease burden worldwide . MI are Despite similar or greater mortality in patients with NSTEMI, their risk compared with that of patients with STEMI is typically perceived by health care providers as lower . FurtherTo date, a direct comparison of survival in NSTEMI versus a suitable comparison population without MI but accounting for comorbidities has not been performed. Such a survival comparison could better define the importance of time-sensitive, myocardium-directed management in NSTEMI. In this work, we analyzed data from a community-based cohort study to test the hypothesis that the hazard of all-cause mortality, controlling for the effects of participant characteristics and common comorbidities, would be higher among those experiencing NSTEMI than among those without an MI.The ARIC (Atherosclerosis Risk in Communities) study cohort was recruited beginning in 1987, during which time each ARIC field center  enrolled a sample of approximately 4,000 individuals ages 45 to 64 years. ARIC methodology is described in detail elsewhere ; brieflyFor our primary analysis, the exposed series comprised participants who experienced an NSTEMI during follow-up. Those who did not experience an MI composed the unexposed series of participants. NSTEMI classification in the ARIC study was based on a hospital discharge diagnosis of probable or definite MI, an equivocal or abnormal cardiac biomarker, and lack of ST-segment elevation.After excluding 647 participants with prevalent MI at baseline, we excluded an additional 192 participants because of insufficient numbers for analyses, such as race/ethnicity other than white or black, along with participants with incident STEMI during follow-up, resulting in a final sample size of 422 participants with NSTEMI during the follow-up period. Among 14,459 eligible participants, 14,037 did not have an incident NSTEMI through 2011 and were eligible for inclusion in the unexposed series. Selecting from the unexposed series with replacement participants without an MI were successfully incidence-density matched to NSTEMI cases as described in detail in the Statistical Analysis section.2) and creatinine (mg/dl) were measured during the medical exam at baseline and were treated continuously in all analyses. Educational attainment was self-reported at baseline and categorized as less than high school or high school or greater. Smoking status was self-reported at baseline and defined as current, former, or never. Medical history was determined via self-report and medical exam at baseline for cancer, diabetes, and lung disease. The presence of left ventricular hypertrophy (LVH) was determined from ECG data. ECG-LVH was defined 2 ways: 1) by Minnesota Codes (3-1 or 3-3) and (any of 4-1 to 4-3 or 5-1 to 5-3), which represent high-amplitude left chest R waves and ST-T changes; and 2) by Cornell voltage criteria. Participants were described as currently taking aspirin, blood pressure–lowering medication, or cholesterol-lowering medications if they reported use within 2 weeks of the baseline exam.Covariates measured at baseline included age, sex, race, and study community; along with selected socioeconomic, clinical, and behavioral characteristics as follows. Common and clinically significant comorbidities were selected a priori for consideration in the analyses. Body mass index , sex, and race/study community  at the time the NSTEMI occurred. Up to 5 participants were matched to each NSTEMI case based on these factors for a total of 608 participants in the unexposed series. All analyses accounted for the matched structure of the data.Survival was assessed from the time of the MI event to death, loss to follow-up, or the end of 2011, whichever came first. Consistent with the incidence-density sampling strategy, the follow-up for the unexposed group began on the same calendar date as did the follow-up for their matched NSTEMI counterparts. We calculated the median time-to-event for each exposure group. The product-limit (Kaplan-Meier) method was used to measure time to death over the course of follow-up. We performed matched Cox proportional hazards regression (frailty models) to estimate hazard ratios (HR) and 95% confidence intervals (95% CI) for all-cause mortality, comparing participants with NSTEMI to the unexposed series. This modeling strategy accounted for the dependence of observations induced by matching.Crude NSTEMI-mortality analyses were conducted, and the influence of covariates were tested in a full multivariable model. Model variable selection was assessed using a p value of <0.05. Analyses were performed with SAS version 9.3  statistical software.We additionally assessed for the effect of NSTEMI, on mortality risk, conditional on surviving 30 days following the MI event. According to these criteria, we included 381 participants with NSTEMI in the exposed group and 185 in the unexposed group.Of the 422 participants who developed NSTEMI, 56% (n = 238) died over a median follow-up of 8.4 years. In contrast, among those who did not experience an MI, 74 (40%) died over a median 9.4 years of follow-up. Kaplan-Meier curves demonstrate a survival difference between NSTEMI and MI-free groups, with the MI-free group experiencing a lower hazard of mortality over the follow-up period (log-rank p < 0.0001) .In the fully adjusted model, the majority of the aforementioned associations remained but were slightly attenuated ; the NSTCommunity participants experiencing incident NSTEMI had a higher hazard of mortality than participants who were MI-free. Whereas those with NSTEMI events had a higher proportion of comorbidities and adverse health behaviors at baseline, NSTEMI itself remained a significant risk factor for mortality after adjustment for these conditions, and this difference persisted over the follow-up period and in analyses conditional on 30-day survival.It may seem obvious that experiencing NSTEMI should confer greater subsequent risk of death compared with not having an MI. However, because this MI-associated mortality hazard persisted despite adjustment for comorbid conditions, myocardial damage itself—the essence of an MI—becomes the lead suspect responsible for downstream mortality. One could then intuit that strategies that reduce myocardial damage in patients with MI should be evaluated to reduce mortality. This logic has translated to remarkably consistent deployment of myocardial damage-limiting approaches for patients with STEMI. For these patients, the adage “time is muscle” has driven changes in health care delivery such as engagement between rural emergency medical services and interventional cardiologists, in-ambulance ECG findings that drive upstream drug delivery, and 24/7/365 in-house staffing of major cardiac catheterization laboratories.Although the study does not directly compare management strategies or MI types, strategies that reduce myocardial damage and ultimately lower mortality in other conditions may warrant evaluation for patients with NSTEMI. A recently published randomized trial for patients with NSTEMI showed lower 30-day death and myocardial damage with immediate (within an average of 1.4 h) versus delayed (median: 61.0 h) angiography . AlthougWhereas prior studies have shown that comorbidities such as diabetes , chronicSimilarly, patients with chronic kidney disease suffering NSTEMI receive less guideline-directed therapy ,19 than Even though our work has many strengths, particularly compared with claims data–based approaches, including careful classification matching of patients with and without NSTEMI to an MI-free population and participant diversity, limitations include reliance on baseline comorbidity information that may have changed by the time of incident NSTEMI in those experiencing MI events. Further investigations including patients whose race and ethnicity extend beyond black and white communities are needed. There remains potential for residual confounding in these analyses that likely biases the observed effect estimates up and away from the null. We also cannot comment on changes in medication between the event and follow-up, which would influence mortality. As there were no systematic cardiac imaging or functional status assessments post-MI, further risk stratification of the post-MI cohort by such data as wall motion abnormalities or heart failure was not feasible. This study spans a significant time period that provides strength to the data in terms of follow-up length; however, the study also spans generations of various different therapies of ischemic heart disease. These limitations extend to both participants with MI and their matched referent group.We have shown that patients with NSTEMI, where comorbid conditions are common, have increased risk of death beyond what can be accounted for by these comorbidities. Further studies of strategies that limit myocardial damage, the central feature of MI, to reduce mortality in patients with NSTEMI are warranted.


Radiologic pleuroparenchymal fibroelastosis (PPFE)-like lesion including pulmonary apical cap can be occasionally observed in clinical settings. However, the significance of radiologic PPFE-like lesion is unclear in connective tissue disease (CTD)-related interstitial lung disease (ILD).A total of 113 patients with CTD-related ILD were enrolled and assessed for radiologic PPFE-like lesion, which was defined as bilateral, upper lobe, and subpleural dense consolidations with or without pleural thickening on chest high-resolution computed tomography. The clinical, radiologic, and pathologic characteristics were evaluated.Radiologic PPFE-like lesion was found in 21 patients (19%) and were relatively frequent in those with systemic sclerosis (6/14: 43%) and primary Sjögren's syndrome (4/14: 29%). Patients with PPFE-like lesion were significantly older, had lower body mass index, higher ratio of residual volume to total lung capacity, and higher complication rate of pneumothorax and/or pneumomediastinum than those without. Twelve of the 21 patients were diagnosed radiologically as usual interstitial pneumonia (UIP) or possible UIP pattern. Two of three patients who underwent surgical lung biopsy of the upper lobes showed UIP on histopathology. Another patient was confirmed to have upper lobe PPFE on autopsy. During the clinical course, progression of the radiologic PPFE-like lesions was observed in 13 of 21 patients. Six patients died  and their PPFE-like lesions were commonly progressive. In the total cohort, our multivariate analysis identified the presence of PPFE-like lesion as a significant risk factor for respiratory death .In patients with CTD-related ILD, radiologic PPFE-like lesion, which may present as not only PPFE but also apical cap and upper lobe subpleural fibrosis commonly due to UIP, was not uncommon and was associated with poor prognosis. Clinicians should be cautious with this radiologic finding, particularly when it is progressive. Pulmonary apical cap is known as a non-morbid finding on chest X-ray or computed tomography (CT). This radiologic term represents an irregular density on the apex of lung that is generally less than 5 mm. The typical pathologic feature is a subpleural fibroelastic scar and is commonly accompanied by thickening of the visceral pleura ,2. ChangConnective tissue diseases (CTDs) are among the important etiologies of interstitial lung disease (ILD). In contrast to idiopathic pulmonary fibrosis (IPF) and idiopathic nonspecific interstitial pneumonia (NSIP), both of which typically show lower lobe-predominant distribution, CTD-related ILD occasionally reveals an upper lobe predominance and, rarely, can coexist with pleural/subpleural disorders, including PPFE –6. BasedIn this retrospective study, we investigated the prevalence of PPFE-like lesion on chest high-resolution CT (HRCT) in patients with CTD-related ILD and compared the clinical, radiologic, and pathologic characteristics between patients with and without the finding. Our aim was to evaluate the clinical significance of radiologic PPFE-like lesion in CTD-related ILD.This study was approved by the institutional review board of Hamamatsu University School of Medicine . Because of the retrospective nature of this study, written informed consent from the subjects was waived.A retrospective database review of patients with ILD at Hamamatsu University Hospital in Japan between 2001 and 2014 identified 133 patients who were diagnosed with CTD-related ILD. Among them, 20 patients were excluded because their follow-up period was shorter than three months. Subsequently, 113 patients with CTD-related ILD were included in the present study. The diagnosis of ILD was based on chest HRCT findings of reticular abnormalities, ground-glass attenuation, or consolidation and clinical exclusion of other pulmonary diseases, such as infectious pneumonia. The time of ILD diagnosis was based on the first HRCT evaluation. Surgical lung biopsy specimens were available for review in 36 patients. Among them, 30 patients had upper lobe specimens in addition to lower lobe specimens. All patients had been diagnosed with CTD before or at the time of ILD diagnosis. The diagnoses of CTDs were based on accepted criteria –12.The medical records were retrospectively reviewed to obtain the following clinical data at the time of ILD diagnosis: demographics; smoking history; laboratory data; pulmonary function test results ; and bronchoalveolar lavage fluid analysis. The patients’ clinical course, including treatment, major respiratory complications, such as acute exacerbation of ILD; respiratory tract infection; lung cancer; and pneumothorax and/or pneumomediastinum, and prognosis, was also recorded. Acute exacerbation of CTD-related ILD was diagnosed, as described previously .HRCT scans of the chest at full inspiration were obtained from all 113 patients at the time of ILD diagnosis. All images were reviewed independently by two chest radiologists with 28 and 14 years of experience, respectively, without knowledge of the clinical and pathologic information of the patients.Radiologic PPFE-like lesion was defined as bilateral, upper lobe, and subpleural dense consolidations with or without pleural thickening on chest HRCT, which corresponds to “consistent with PPFE on radiology” proposed by Reddy et al. . The posHistologic sections of lung specimens, from 36 patients by surgical lung biopsy and from 1 patient by autopsy, were stained with hematoxylin–eosin and elastic van Gieson. All slides were reviewed by an experienced lung pathologist (TVC) who was not aware of the clinical and radiologic findings.For the surgical lung biopsies, the following pathologic findings were semi-quantitatively graded as 0 (absent), 1 (mild), 2 (moderate), or 3 (severe): interstitial fibrosis; fibroblastic foci; lymphoplasmacytic infiltration; lymphoid follicles with germinal center; and emphysema. The presence or absence of a finding of PPFE, defined as upper-zone pleural fibrosis with subjacent intra-alveolar fibrosis accompanied by alveolar septal elastosis , was alData were described as number (percentage) or median (interquartile range), unless otherwise stated. Group comparisons were performed using Chi-square test, Fisher’s exact test, or Mann–Whitney U test, as appropriate. Overall survival was defined as the time from the date of ILD diagnosis to the date of censoring or death. Patients were censored if they remained alive until June 30, 2015 or if they dropped out of the follow-ups. The survival curves were generated by the Kaplan–Meier method and were compared by log-rank test. Cox proportional hazard model was used to evaluate the prognostic impact of radiologic PPFE-like lesion, in conjunction with other well-known prognostic factors in ILDs, including age, gender, % predicted FVC, % predicted DLCO, and UIP compatibility on HRCT ,20. All 2, % predicted FVC, and % predicted DLCO between the groups, but the ratio of RV to TLC was significantly higher at a median value of 44% (interquartile range: 37%–47%) in patients with PPFE-like lesion.The baseline patient characteristics upon diagnosis of CTD-related ILD and comparison of patients based on the presence of radiologic PPFE-like lesion are summarized in The radiologic findings at the time of CTD-related ILD diagnosis are shown in The clinical course of the patients according to the presence or absence of radiologic PPFE-like lesion is compared on In 21 patients with radiologic PPFE-like lesion, the pulmonary function data one year (between 9 months and 15 months) after the diagnosis of CTD-related ILD were available in 16 patients. Interestingly, patients with “progressive” PPFE-like lesion (n = 10) commonly showed the deterioration of their pulmonary function . In contrast, most patients with “stable” PPFE-like lesion (n = 6) maintained or improved their function .In the total cohort, the median follow-up period was 60.7 months (interquartile range: 31.0–99.2 months). During the period, there were 6 and 17 deaths, respectively, among patients with and without radiologic PPFE-like lesion. No patients received lung transplantation. The causes of death were variable in patients without radiologic PPFE-like lesion. On the other hand, in the six patients with the lesion, all the causes of death were respiratory diseases, such as worsening of chronic respiratory failure (n = 3), acute exacerbation of ILD (n = 2), and respiratory tract infection (n = 1). In 5 of the 6 non-surviving patients, the PPFE-like lesions were “progressive” .The survival curves are shown in In our cohort of CTD-rerated ILD, radiologic PPFE-like lesion was detected in as many as 19% of patients, most commonly in those with radiologic and/or pathologic UIP pattern. Intriguingly, despite the possibility that our criteria might have included incidental apical caps, the presence of PPFE-like lesion was identified as an independent poor prognostic factor. To the best of our knowledge, our result is the first to delineate the clinical significance of radiologic PPFE-like lesion in CTD-related ILD.Pulmonary apical cap is not a rare radiologic finding and the prevalence in the general population was reported as around 10%, more predominantly in older individuals . Of notePPFE is a distinct pathologic pattern and the idiopathic type has been recently recognized as a rare form of IIPs ,18,21,22In our CTD-related ILD patients, the presence of radiologic PPFE-like lesion was a significant risk factor for death due to respiratory diseases. One of the explanations may be the predominance of UIP pattern, which is a well-known poor prognostic determinant, particularly in IIPs and rheumatoid arthritis-related ILD ,24. GeneThe major limitation of the present study was that it was a small, single-center retrospective study. In particular, the small number of subjects who underwent pathologic evaluation made it difficult to fully evaluate the correlation between the radiologic and pathologic findings. The distinction between radiologic PPFE-like lesion with clinical significance and pure apical caps remains unclear, if at all possible. It may be helpful to confirm disease progression by serial radiologic assessment.Radiologic PPFE-like lesion, which may present as not only PPFE but also apical cap and upper lobe subpleural fibrosis commonly due to UIP, was not uncommon and was associated with worse prognosis in patients with CTD-related ILD.S1 Table(DOCX)Click here for additional data file.


Despite the advances in the treatment of obstructive sleep apnea (OSA) with mandibular advancement appliances (MAA), their effectiveness is dependent on the patients’ compliance. Our aims were to evaluate the long-term adherence to MAA therapy and patients’ experiences of the treatment in OSA.Sixty-nine patients  were included in the study. The subjects were mild (56%) and moderate (44%) OSA patients who had been treated using MAA at least 4 years prior to the study. A phone survey was used to determine the demographic characteristics of the patients, as well as to assess self-reported adherence to therapy, subjective long-term effectiveness, and patient experiences with the appliance. Descriptive statistics, Pearson Chi-square test, and independent samples t-test were used for data analysis.p<0.05). The most common reasons reported by patients were inability to adapt to the appliance (62%) and pain in the temporomandibular joint (38%). The most common factors associated with continued usage were effectiveness (100%) and ease of use (64%).Only 22 (32%) patients reported using the appliance regularly. Most of the non-adherent patients had stopped using their appliances in the first year (55%). The mean duration of appliance use was 33.5 months (Median: 12 months). No significant differences in appliance type, OSA severity, educational level, gender, marital status, income status, employment status or place of residence existed between adherent and non-adherent subjects. Adherent subjects were significantly younger than non-adherent subjects (Age: 50.6 ± 11.9 versus 56.1 ± 9.9, The overall long-term nonadherence to MAA therapy in mild-to moderate OSA patients was high suggesting that barriers to MAA therapy adherence should be prevented to increase the efficiency of oral appliance treatment in OSA and achieve better outcomes for this disease. Key words:Obstructive sleep apnea, mandibular advancement, therapy, oral appliance, long-term compliance. Obstructive sleep apnea (OSA) is a highly prevalent sleep-related breathing disorder characterized by periods of recurrent cessation of breathing caused by partial or complete collapse of the upper airway. Untreated OSA is associated with cardiovascular disorders, cerebrovascular disease, and cognitive dysfunction . The golMandibular advancement appliances (MAA) act by protruding the mandible and increasing the upper airway size and they are the most commonly used type of OA’s in the treatment of OSA . Their uDespite the advances in the treatment of sleep apnea with oral appliances, their effectiveness is dependent on the patients’ adherence. Adherence to a medical regimen is defined as the successful adoption of a treatment program . AccordiAfter obtaining the institutional ethics committee approval, 77 patients  who were successfully treated with an MAA at our clinic between 2005 and 2012 (at least 4 years prior to the study) were contacted by phone. These patients had been diagnosed with sleep apnea at the Department of Otorhinolaryngology or the Department of Chest Diseases using an overnight polysomnography. Their disease severity had been classified as mild  or moderate  , and theA phone survey was used to determine the demographic characteristics of the patients, as well as to assess self-reported adherence to therapy, subjective long-term effectiveness, and patient experiences with the appliance. Patients who stopped using their appliances were classified as ‘’non-adherent’’, while those who were still using their MAA’s were classified as ‘’adherent’’.-Statistical analysisp < .05 (two-tailed).Statistical analyses were performed using SPSS, Version 21.0 . Demographic data were analyzed using descriptive statistics which included the mean, standard deviation and percentage distribution. Categorical variables were compared between adherent and non-adherent groups using Pearson Chi-square test. For continuous variables independent samples t-test was used. Statistical significance was defined as Demographic characteristics of the patients are presented in p < .05).Only 22 (32%) subjects reported using the appliance regularly. Most of the non-adherent patients had stopped using their appliances in the first year (55%), 10% in the second year and another 15% in the fourth year  were treated with a monobloc appliance, which was the most frequent type of MAA at the time. Some studies report that monobloc appliances are as effective as bibloc appliances although the effect remains uncertain . Patientet al.  found th (et al.  found th (et al.  have notet al. (et al. (Almeida et al.  reported (et al.  found thOur research has some limitations. The first one is the use of subjective compliance data to determine adherence rates. Despite the limitations of this method, it is still commonly used in adherence studies. Furthermore, a high agreement between objective and subjective compliance data in oral appliance therapy has been reported in literature . NeverthIn conclusion, long-term adherence rates in MAA therapy of mild-to-moderate OSA patients is lower than those reported in short-term studies and younger patients show higher compliance rates. Since oral appliance therapy is the first-line of treatment for a number of these patients, factors affecting adherence should be explored in future studies. Furthermore, interventions aimed at improving adherence should be designed to ensure the successful treatment of this chronic disease.


Lycopersicon esculentum) is a fruit with diverse bioactive nutrients that is consumed worldwide. In this study, we investigated the cognition-enhancing effect of tomato ethanolic extracts (TEE) in aged mice. Six weeks of oral TEE administration in 12-month-old aged mice significantly increased their exploration time of novel objects when compared to vehicle-treated mice. The TEE supplement increased doublecortin (DCX)-positive cells and postsynaptic density-95 (PSD95) expression in mice hippocampus. Moreover, we found an increased expression of brain-derived neurotrophic factor (BDNF) and subsequently-activated extracellular-signal-regulated kinase (ERK)/cAMP response element binding (CREB) signaling pathway in the TEE-supplemented mice hippocampus. In conclusion, the oral administration of TEE exhibits a cognition-enhancing effect, and the putative underlying mechanism is the induction of BDNF signaling-mediated proliferation and synapse formation in the hippocampus. These findings indicate that TEE could be a candidate for treatment of age-related memory impairment and neurodegenerative disorders.A decrease in adult neurogenesis is associated with the aging process, and this decrease is closely related to memory impairment. Tomato ( Neurogenesis is a process that consists of the generation of neurons from neural stem cells and progenitor cells. The subgranular zone (SGZ) in the hippocampus is a major part of the brain involved in adult neurogenesis . NeurogePrevious studies have demonstrated the neurogenic and cognition-enhancing activities of several plant-derived agents ,7. The bLycopersicon esculentum) is consumed worldwide as a raw fruit or in processed products, and it is an important dietary source of nutrients such as polyphenols, flavonoids and several antioxidants. Diverse active compounds in tomato have shown pharmacological efficacy in several types of cancer [Tomato  test after administration of tomato ethanolic extracts (TEE) for six weeks and investigated the expression of BDNF and the relevant signaling pathway in the hippocampus.The antibodies used in this study were as follows; mouse monoclonal antibodies against postsynaptic density-95 , goat polyclonal antibodies against β-actin , goat polyclonal antibodies against doublecortin , rabbit polyclonal antibodies against phospho-ERK  and polyclonal antibodies against total ERK .TEE was prepared by the Korea Food Research Institute . Tomatoes were sliced, shade dried, powdered, and sequentially extracted with 50% alcohol twice for 3 h each. The extract was filtered, condensed by a vacuum, and freeze-dried at −40 °C under a reduced pressure (yield: 46.18% of dry weight).ad libitum and were acclimated for one week prior to the study. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Center for Phenogenomics Animal Research, Woojung BSC . The aged mice were randomly divided into two equivalent groups, one administered vehicle and the other TEE. The animals were orally fed with a dose of 400 mg/kg using a feeding needle once daily for six weeks and were sacrificed six hours after the last administration. For vehicle-treated young and aged mice, the same volume  of 0.5% carboxymethyl cellulose-sodium solution was administered once daily for six weeks  were purchased from Orient Bio . The animals were allowed to feed ix weeks A. Each gTo investigate whether TEE has a cognition-enhancing effect, TEE was orally administered to 12-month-old aged mice for 6 weeks, and the NOR test, a non-forced recognition memory test, was performed using a modified method . BrieflyThe mice were anesthetized with an intramuscular injection of Zoletil  and xylazine solution (3:1 ratio) and were transcardially perfused with 0.9% normal saline. The brains were carefully dissected and were separated into two hemispheres. For the biochemical analyses, the hippocampus was removed from the left hemisphere, snap-frozen in liquid nitrogen, and stored at −80 °C. The right hemisphere was fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) overnight at 4 °C and was equilibrated in 30% sucrose. The brain tissues were cut into sequential coronal sections (40 μm thick) with a cryostat  and were individually collected in 24-multiwell culture plates.Immunostaining for DCX was performed using the free-floating technique. One series was randomly selected and stained using antibodies against DCX . The free-floating slices were incubated for two days at 4 °C with the primary antibodies in a diluent buffer comprising 1% bovine serum albumin (BSA) and 1% Triton X-100 in 0.1 M phosphate buffer. Subsequently, the sections were incubated for 24 h at 4 °C with biotinylated rabbit anti-goat IgG antibody . The sections were then incubated with a Vector ABC kit . DCX-positive cells were visualized with 3,3′-diaminobenzidine (DAB) , and the images were taken with a Leica DM5500B microscope (Leica) . To quantify the total number of DCX-positive cells in the SGZ, all sections were coded, and cell counting was performed with the examiner blinded to the group allocation.g for 15 min at 4 °C, and the supernatants were used for analysis. ELISA was performed using the Corticosterone ELISA kit  to measure the endogenous corticosterone and the BDNF Emax Immunoassay System , to measure the BDNF levels in the hippocampus. All procedures were carried out according to the manufacturer’s instructions. The corticosterone and BDNF levels were quantified at 450 nm using an ELISA reader  and were analyzed by a standard curve.The hippocampus was homogenized in lysis buffer , 10 μg/mL aprotinin, 1 μg/mL leupeptin, and 0.5 mM sodium orthovanadate; pH 7.2) and then centrifuged at 15,000× A western blot analysis was performed as previously described . The prot-tests, and a p-value of less than 0.05 was considered to be statistically significant. The data from the immunohistochemistry, western blot and ELISA were analyzed using one-way ANOVA with Newman-Kuels post hoc tests for the multiple comparisons tests. The results are expressed as means ± standard error of the mean (SEM), and the statistical analyses were performed using the SPSS 22.0 software .The behavioral data were analyzed using unpaired Student’s A total of eighteen mice (nine mice/group) completed the study. To examine whether TEE administration was associated with a change in the body weight of mice, we measured the body weight every week. Six weeks of TEE treatment did not influence the body weight of the mice when compared to the vehicle-treated group C.During the training phase, there were no statistical differences in the percentage of time spent exploring two identical objects A. During+ cells in the DG in aged mice (12 months of age) compared to young (8-week-old) mice . However, the mice supplemented with TEE showed a significant increase (1.6-fold) in the number of DCX+ cells in DG , as shown in The above results indicated that the TEE treatment enhanced cognitive function in aged mice. To investigate whether the TEE treatment affected hippocampal neurogenesis, the overall cell proliferation in the SGZ of the dentate gyrus (DG) was assessed via immunohistochemistry for DCX, a marker for immature neurons. We found a significant reduction (14.8-fold) in the number of DCXTo investigate the possible mechanisms of cognition enhancement and neurogenesis for TEE, we analyzed the corticosterone and BDNF levels in hippocampus via ELISA. Although BDNF is significantly decreased in the hippocampus of aged mice when compared to young mice, TEE administration significantly increased the hippocampal BDNF protein by 32.6% ± 6.7% in aged mice A. There Chronic exposure to corticosterone down-regulates the BDNF expression, and decreases neurogenesis in the hippocampus . In addiThe decline in adult neurogenesis and subsequent memory impairment is a major event associated with aging that is referred to as neurodegeneration , and theOver the past decade, natural ingredients from plants, vegetables, and fruits have been used as sources of pharmaceutical agents. For example, flavonoids from many kinds of plants have diverse pharmacological activities, including anti-oxidant, anti-inflammatory and anti-cancer effects ,26, and The cognitive function was evaluated using the NOR test, which is one of the most frequently used methods for assessing memory alterations in various subfields within neuroscience . During To support our behavioral test, we investigated the morphological changes and proliferation of neuronal cells in the DG, as determined via DCX immunostaining. DCX plays a critical role in promoting microtubule polymerization, and it is expressed in neuronal precursor cells and immature neurons . In geneOral TEE treatment significantly increased the age-related reduction in BDNF expression in the hippocampus. To confirm the effect of TEE, we investigated whether TEE enhances BDNF production in brain tissues isolated from young mice. ICR mice  were divided two groups; vehicle-treated group and TEE-treated group. The mice were orally administered with 400 mg/kg of TEE every day for two weeks. As a result, the mRNA level of BDNF in TEE-treated group was significantly increased (1.89-fold) compared with vehicle-treated group , as shown in Corticosterone levels in hippocampus were significantly decreased in the TEE-treated mice. Corticosterone is known as a stress hormone, and it is also associated with cognitive impairment . Recent BDNF is a neurotrophic factor involved in neurogenesis, the modulation of synaptic plasticity, and the release of neurotransmitters . BDNF-trLycopene and rutin have been known as active compounds of tomato. To reveal active components in TEE, we analyzed components in TEE using HPLC and LC/MS. However, high concentrated peaks were not found, which are able to represent functionality of TEE. In the case of lycopene, it was included at 0.0001478% in TEE (data not shown). In addition, rutin was also included at 0.00696% in TEE. These result indicated that polyphenols, flavonoids, and non-polar components reported as functional components in tomatoes showed low possibilities of being active components in the functionality of TEE. Therefore, we suggest that the enhancing effect of TEE on cognitive function via BDNF production might be derived from poly/oligo-saccharides because TEE (50% ethanolic extract of tomato) contained various hydrophilic saccharides ,44.Animal models with cognitive impairment have been used as a tool to evaluate cognitive functions since these reflect the complex interactions among diverse neural systems. Several new mouse models are now available with cognitive impairment induced by pharmacologic, toxicological, and genetic methods . AlthougIn this study, we showed that oral administration of TEE for 6 weeks enhanced cognition in aged mice. The TEE supplement improved the age-related reduction of hippocampal neurogenesis, increased BDNF production, and increased the levels of phosphorylated ERK and CREB in the hippocampus. Taken together, the cognition-enhancing effect of TEE might be attributed to increased neurogenesis and synapse formation in the hippocampus via activation of the BDNF signaling pathway. These results suggest that TEE can be a potential candidate for treating age-related memory impairment and neurodegenerative disorders. Furthermore, we suggest that increased consumption of tomato  would contribute to protect against brain aging.


Surrogates of cortical information exchange in high-frequency gamma are increased in association with accelerated recovery from anesthesia. This finding adds evidence suggesting a functional significance of high-gamma information transfer in consciousness.Recent data from our laboratory demonstrate that high-frequency gamma connectivity across the cortex is present during consciousness and depressed during unconsciousness. However, these data were derived from static and well-defined states of arousal rather than during transitions that would suggest functional relevance. We also recently found that subanesthetic ketamine administered during isoflurane anesthesia accelerates recovery upon discontinuation of the primary anesthetic and increases gamma power during emergence. In the current study we re-analyzed electroencephalogram (EEG) data to test the hypothesis that functional cortical connectivity between anterior and posterior cortical regions would be increased during accelerated recovery induced by ketamine when compared to saline-treated controls. Rodents were instrumented with intracranial EEG electrodes and general anesthesia was induced with isoflurane anesthesia. After 37.5 min of continuous isoflurane anesthesia, a subanesthetic dose of ketamine  was administered, with evidence of a 44% reduction in emergence time. In this study, we analyzed gamma and theta coherence  and normalized symbolic transfer entropy  between frontal and parietal cortices during various levels of consciousness, with a focus on emergence from isoflurane anesthesia. During accelerated emergence in the ketamine-treated group, there was increased frontal-parietal coherence { There is convergent evidence from multiple studies involving multiple species, multiple neuroimaging modalities, multiple analytic techniques, and diverse drug classes suggesting that anesthetic-induced unconsciousness is characterized by a functional fragmentation of cortical and thalamocortical networks  were randomly divided into ketamine and saline groups. At least one week before the experiment, animals underwent head cap surgery. For the purposes of this surgery, general anesthesia was induced with 4% isoflurane , and then reduced to 2.5%. Rats received a single dose of the non-steroidal anti-inflammatory drug carprofen . Craniotomy was then performed and six electrodes made out of stainless steel wire  were placed at the following stereotaxic coordinates to record EEG: one frontal at 3.0 mm anterior to bregma and 2.5 lateral to the midline , two parietal at 4 mm posterior to bregma and 2.5 mm lateral to midline on both sides, two occipital at 8 mm posterior to bregma and 2.5 mm lateral to midline on both sides and one on the nasal commissure as the reference electrode. In addition, rats had two electromyography electrodes placed into the neck muscle to evaluate muscle movement during emergence. Animals were then allowed to recover from surgery for 7 days.As noted, we re-analyzed EEG data from a previously reported study  and filtered . Data were digitized and recorded at a sampling rate of 1,000 Hz using AcqKnowledge software . The raw EEG signals from ipsilateral frontal and parietal channels were exported into MATLAB  and downsampled to 500 Hz  for further analysis. By visual inspection and spectral analysis of the EEG signals, one ketamine-treated rat and three saline-treated rats were excluded because of bad signal quality. Thus, the sample sizes in subsequent analysis were N = 20) of surrogate data were generated by randomly shuffling one of the two signals, while keeping the other signal unchanged . Specifically, the two time series of 10-s were divided into 2-s sub-windows with 80% overlap, each sub-window was multiplied with a Hamming window, and the coherence was estimated from the cross-spectra and auto-spectra of two series using Welch's averaged, modified periodogram method. To mitigate the potential bias of coherence, a series , low-gamma (25–50 Hz), med-gamma (70–110 Hz), high-gamma (110–160 Hz). We chose these general bandwidths because of their relevance to arousal states based on our recent study of neural correlates of wakefulness in rats  to assess directed connectivity. NSTE is an information-theoretic measure, and our previous studies have validated its use to measure cortical connectivity changes in humans , time delay (τ), and transfer time (δ). We filtered the frontal and parietal EEG signals into four indicated frequency bands  and segmented the filtered data into non-overlapped 10-sec windows. For all windows, we followed previous studies  were estimated and plotted. Statistical comparisons were analyzed using linear mixed models (LMM), to test (1) the difference between ketamine-treated (n = 9) and saline-treated (n = 7) rats, (2) the changes between the six studied epochs within the ketamine- or saline-treated groups, and (3) the difference between feedback and feedforward connectivity. LMM analysis offers more flexibility in dealing with unequal numbers of rats and allowed us to take into account the variability between rats by including a random effect associated with the intercept for each rat. For the model of coherence values, the fixed effects included group, studied epoch, and the interaction between them. For the models of directed connectivity, the fixed effects included group, studied epoch, direction, and the interaction between every pair of factors. We used restricted maximum likelihood estimation and diagonal covariance structure with heterogeneous variances and zero correlation between elements. For all statistical comparisons, the uncorrected p-value and the 95% CI of the difference were reported. A p < 0.05 was considered statistically significant. To enhance readability of the figures, only significant levels are provided, while the exact p-value and 95% CI are provided in a tabular format in the supplementary material.Statistical analyses were conducted in consultation with the Center for Statistical Consultation and Research at the University of Michigan. All EEG-derived frontal-parietal coherence and feedback/feedforward connectivity values were exported to IBM SPSS Statistics version 24.0 for Windows  and GraphPad Prism version 7.00 for Windows . The descriptive statistics ] ; feedforward: p < 0.001, 0.009–0.025 (95% CI)]; there was no significant difference between feedback and feedforward connectivity in either ketamine- or saline-treated rats . In addition, ketamine-treated rats showed increased theta feedback and feedforward connectivity at emergence  and even before the discontinuation of isoflurane . Consistent with frontal-parietal coherence findings, both ketamine- and saline-treated rats achieved the highest level of high-gamma connectivity at emergence, while mid-gamma connectivity showed gradual increases after RORR, and theta connectivity remained at low levels during recovery as compared to baseline  and directional (NSTE) connectivity and was present in both theta and high-gamma bandwidths. We previously demonstrated that theta connectivity (measured by both coherence and NSTE) is elevated during both wakefulness and rapid-eye-movement (REM) sleep  to a primary inhaled anesthetic (isoflurane) was the causal agent that accelerated recovery and enhanced high-gamma connectivity. The administration of subanesthetic ketamine causes “paradoxical emergence” because it deepens the intra-anesthetic state  while reducing the time to emergence once the isoflurane is discontinued (Hambrecht-Wiedbusch et al., There are limitations to this study. In terms of the scientific implications, the current methodology does not allow us to distinguish whether high-gamma connectivity is a cause, correlate, prerequisite, or consequence of conscious experience (Aru et al., In conclusion, accelerated recovery of consciousness after general anesthesia is associated with enhanced connectivity in the high-gamma bandwidth. This supports the hypothesis that coherent patterns of high-frequency communication across the cortex are functionally important for consciousness but further investigation is required to establish causal relationships.GM conceived of the study; VH performed experiments; DL performed analysis; GM and DL wrote the manuscript; all authors critically reviewed and approved manuscript.Supported by grant R01GM111293 from the National Institutes of Health, Bethesda, Maryland, USA and the Department of Anesthesiology at the University of Michigan, Ann Arbor, Michigan, USA.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SM and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Previous studies revealed high incidence of acquired N-glycosylation sites acquired N-glycosylation sites in RNA transcripts encoding immunoglobulin heavy variable region (IGHV) 3 genes from parotid glands of primary Sjögren’s syndrome (pSS) patients. In this study, next generation sequencing was used to study the extent of ac-Nglycs among clonally expanded cells from all IGVH families in the salivary glands of pSS patients. RNA was isolated from parotid gland biopsies of five pSS patients and five non-pSS sicca controls. IGHV sequences covering all functional IGHV genes were amplified, sequenced, and analyzed. Each biopsy recovered 1,800–4,000 unique IGHV sequences. No difference in IGHV gene usage was observed between pSS and non-pSS sequences. Clonally related sequences with more than 0.3% of the total number of sequences per patient were referred to as dominant clone. Overall, 70 dominant clones were found in pSS biopsies, compared to 15 in non-pSS. No difference in percentage mutation in dominant clone-derived IGHV sequences was seen between pSS and non-pSS. In pSS, no evidence for antigen-driven selection in dominant clones was found. We observed a significantly higher amount of ac-Nglycs among pSS dominant clone-derived sequences compared to non-pSS. Ac-Nglycs were, however, not restricted to dominant clones or IGHV gene. Most ac-Nglycs were detected in the framework 3 region. No stereotypic rheumatoid factor rearrangements were found in dominant clones. Lineage tree analysis showed in four pSS patients, but not in non-pSS, the presence of the germline sequence from a dominant clone. Presence of germline sequence and mutated IGHV sequences in the same dominant clone provide evidence that this clone originated from a naïve B-cell recruited into the parotid gland to expand and differentiate locally into plasma cells. The increased presence of ac-Nglycs in IGHV sequences, due to somatic hypermutation, might provide B-cells an escape mechanism to survive during immune response. We speculate that glycosylation of the B-cell receptor makes the cell sensitive to environmental lectin signals to contribute to aberrant B-cell selection in pSS parotid glands. Primary Sjögren’s syndrome (pSS) is clinically characterized by complaints of dry mouth and dry eyes (sicca complaints), which are associated with the presence of periductal lymphoid infiltrates in the salivary and lacrimal glands. From a pathogenic point of view, B-cell hyperactivity is a hallmark of the disease . This isThe periductal infiltrate of the salivary glands harbors many B-cells, which even can form ectopic germinal centers (GCs), and there is a profound increase in the number of IgG plasma cells . Mucosa-In our previous study , the anaOur previous finding that ac-Nglycs are enriched in IGHV3 transcripts from clonal expansions of Ig-producing cells in parotid glands of pSS patients may point toward a selective advantage for B-cells to expand and survive. A question that arises is whether these ac-Nglycs are unique for IGHV3 genes or are also seen in IGHV genes from other IGHV families. Furthermore, it remains to be shown whether ac-Nglycs are restricted to clonally expanded B-cells and plasma cells, and to what extent BCRs with ac-Nglycs encode for autoantibodies, such as stereotypic RF. Finally, it has to be unraveled whether naïve and/or memory B-cells proliferate and expand to form clones within the microenvironment of the salivary gland tissue of pSS patients. Here, we utilize next-generation sequencing (NGS) to address the above issues regarding clonal expansion of Ig-producing cells in pSS patients.Parotid biopsies of five pSS patients fulfilling the ACR-EULAR  criteriaIn addition, five parotid biopsies of patients with sicca complaints were included as non-pSS sicca controls . These non-pSS sicca controls have the same subjective complaints of dry mouth and dry eyes as pSS patients, but they do not fulfill the ACR-EULAR criteria for pSS. A biopsy of the parotid gland under local anesthesia was taken as part of the clinical diagnostic work-up for pSS . HistopaParotid biopsies were originally collected for diagnostic purposes. Usage of these biopsies for research purposes was obtained on written informed consent with approval from the medical ethical committee of the University Medical Center Groningen, Groningen, the Netherlands.Parotid gland biopsies were snap-frozen in liquid nitrogen after surgery and cryo-preserved at −80°C until use. RNA was isolated from parotid gland samples using a polytron tissue homogenizer  in the presence of STAT60 RNA reagent  according to the manufacturer’s protocol. After isolation RNA was purified using the RNeasy Mini System [Clean-up-protocol ]. RNA quality was checked using the Bioanalyzer 2100 system (Agilent) and quantified using the Qubit1.0-platform . cDNA was synthesized using Superscript RT-III and oligo-dT primers according to the manufacturer’s protocol .Linear amplification of the IGHV genes was based on the protocol used previously , 30 usinSamples were again purified, quantified, prepared for sequencing according to the manual for amplicon sequencing, and sequenced on a Roche Genome Sequencer FLX (titanium platform).The bioinformatics pipeline used to obtain the IGHV sequences was done by performing Multiplex Identifier sorting, identification of gene segments, and removal of artifacts.The IGHV sequences were analyzed for IGHV gene usage, rearrangement, and mutations by aligning them with the human Ig set of the IMGT reference directory.Immunoglobulin heavy variable region sequences with identical nucleotide sequences at the CDR3, and shared the same VH germline gene, were considered as clonally related. Together clonally related sequences form a “clone.” Clones were considered dominant when the number of clonally related sequences was ≥0.3% of the total number of unique recovered sequences per patient (arbitrarily chosen) of the total number of productive sequences obtained from a single biopsy.To examine whether the IGHV sequences encode for stereotypic RF BCRs, we analyzed the IGHV sequences for the presence of stereotypic RF VH/JH rearrangements, combined with VH-CDR3 AA sequence homology to stereotypic RF VH-CDR3 AA sequences , 31, 32.To gain understanding of the origin of the clonal expansion of B-cells in the parotid gland, lineage trees were created from clonally related IGHV sequences using the algorithm of the IgTree© . BrieflySelection pressure analysis of the IGHV sequences belonging to a dominant clone was studied using the online Bayesian Estimation of Antigen-Driven Selection in Immunoglobulin Sequences (BASELINe) program. This program uses statistical algorithms based on analysis of somatic mutation patterns of the IGHV sequences to predict the selection pressure which shape the IGHV repertoire of Ig-producing cells. The distribution of replacements versus silent (R/S) mutations, in both CDRs and FWRs is counted separately and compared against the expected frequency. BASELINe aggregates the selection strength of different sequences within a single experimental group and to compare the selection pressures between different experimental groups .Ac-Nglycs were predicted on the basis of the translated AA sequences of the IGHV sequences (including the CDR3 region) using the NetN-glyc 1.0 online server.U test. p Values <0.05 were considered as statistically significant.All statistical analyses were performed using GraphPad Prism software (version 3.0). Statistical comparisons of data from pSS patients and non-pSS sicca controls were carried out using Mann–Whitney A total of 15.756 unique IGHV sequences were collected from five parotid biopsies of pSS patients (1.815–4.039 per biopsy) and 11.834 unique IGHV sequences in the 4 biopsies of non-pSS sicca controls (2.673–3.191 per biopsy).p = 0.0268) more frequently used in non-pSS sicca  than in pSS  biopsies. The CDR3 lengths of the IGHV sequences between the two patient groups were also similar (data not shown).No significant differences in the usage of various VH genes among the IGHV sequences derived from pSS and non-pSS sicca biopsies were observed except for VH3-72 Figure . The latp = 0.0159) higher percentage of germline encoded IGHV sequences was found among sequences obtained from the parotid gland of pSS patients  lower numbers of mutations than the non-pSS sicca sequences: 6.5% (IQR: 5.17–9.48% per IGHV sequence) and 7.3% (IQR: 4.17–8.62% per IGHV sequence), respectively.Next, we analyzed the presence of somatic hypermutations among the IGHV sequences amplified from cDNA of parotid glands of individual pSS patients and non-pSS sicca controls. IGHV sequences with ≥1 nucleotide difference with the closest reference germline gene were considered to be mutated as a consequence of somatic hypermutation. A significant (p = 0.0159) . Analysis of IGHV sequences derived from the dominant clones of the non-pSS sicca controls showed positive selection pressures in the CDRs (CDR1 and CDR2) and negative selection pressures in the FWRs  Figure . Such a p = 0.0159) in pSS patients, compared to non-pSS sicca controls  of N-glycosylated IGHV sequences in pSS was observed , 56 sequences by VH4 genes (31%), and 3 sequences by VH5 genes (2%) in line with the different number of IGVH genes in the respective IGVH gene families. In non-pSS sicca controls, two of the IGHV sequences derived from dominant clones containing ac-Nglycs were encoded by VH3 genes and one by VH4. The majority of ac-Nglycs in pSS patients were the result of a replacement mutation in the FWR3 region: 115 out of 189 (61%) ac-Nglycs were located in the FWR3 region. The remaining 74 (39%) ac-Nglycs were located in the CDR compartment (CDR1 or CDR2). No ac-Nglycs were observed in FWR1 or FWR2. In the IGHV sequences derived from non-pSS sicca controls, all three ac-Nglycs were located in the CDRs  of the 84 ac-Nglycs in IGHV3 sequences were generated by an AA change at position 84 in the FWR3 region. There was also a high frequency (39%) of 22 ac-Nglycs in the FWR3 of IGHV4 sequences at AA position 90.These data suggest the presence of hot-spots for ac-Nglycs in certain IGHV genes, particularly in the FWR3 region. At these sites, single change in nucleotide can lead to ac-Nglycs due to the composition of the flanking nucleotides.To study clonal evolution in relation to ac-Nglycs, the IGHV sequences belonging to a dominant clone were implemented into the algorithm of IgTree©. Mutations leading to new ac-Nglycs could be found at every level of the lineage tree constructed from the clonally related IGHV sequences of the various dominant clones from the pSS patients; an example of a lineage tree of a dominant clone-derived from a parotid gland of a pSS patient is shown in Figure Analysis of the lineage trees also showed that in four out five pSS patients, a clone was found that comprised the original, unmutated, germline sequence among the observed IGHV sequences. Such clones were not observed in non-pSS sicca controls. The presence of both the germline root sequence and mutated IGHV sequences within the same dominant clone provide evidence that this clone developed from a naïve B-cell that was recruited into the parotid gland where it underwent local expansion and somatic hypermutation. We found no differences in trunk length and path length indicating that there is no difference in affinity and selection threshold in the lineage trees generated from dominant clones of pSS compared to non-pSS sicca (data not shown). Based on these two parameters, we could not discriminate in the diversification history between pSS and non-pSS.Parotid gland MALT lymphomas frequently express BCRs, characterized by a striking AA sequence homology to the IGHV-CDR3 of stereotypic RFs. These RFs are encoded by typical combinations of restricted IGHV and IG kappa V gene rearrangements. Here, we focused on the IGHV rearrangements. Five groups of somatically mutated stereotypic RF BCRs have been identified , 31, 32.With NGS technology, we showed, in agreement with others, that large numbers of Ig-producing clones are present in the glandular tissue , 36, 37 Little is known about the origin of the large number of B-cell clones found in parotid glands of pSS patients. One possible explanation is that these clones arise from CD27+ memory B-cells that enter the salivary glands, where they subsequently clonally expand , 38. We An obvious site for this expansion and somatic hypermutation are the (ectopic) GCs that can be present in the periductal infiltrates of the salivary glands. Similar to regular GCs in peripheral lymphoid organs, these ectopic GCs are composed of activated B-cells, T-cells, and follicular dendritic cell networks , 39–41. We did not find indications for signs of antigen selection in the dominant clones in pSS patients. However, we observed a significantly higher incidence of ac-Nglycs in the IGHV sequences derived from the dominant clones in parotid gland tissue from pSS patients compared to non-pSS sicca controls. We observed that the majority of the ac-Nglycs were present in certain hot-spots, in particular in the FWR3 region of IGHV3 and IGHV4 sequences. A single nucleotide change at this spot during the somatic hypermutation process could already result in ac-Nglycs. The increased frequency of ac-Nglycs is, however, not restricted for IGHV sequences of the dominant clones. The higher incidence of ac-Nglycs seems to be a common phenomenon in the total pool of IGHV sequences obtained from the salivary gland biopsies of pSS patients Figure . Moreovein vitro compared to the non-glycosylated ANCA IgG. However, the role of Fc glycosylation cannot completely be excluded in this assay (Fc-glycosylated antibodies have been found in anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis (AAV), SLE, pSS, and RA. Fc-glycosylation patterns were associated with disease activity and severity and outcome –49. In Ris assay . Sometimis assay , 50. Of in vitro to mannosylated Igs of follicular lymphoma cells to trigger BCR-mediated signaling events (Ca2+flux). Binding of lectins to glycosylated Igs was presumed to lead to survival and even to expansion of lymphoma cells. Lectins are widespread and are both expressed by many cell types, such as T-cells, NK cells, macrophages, dendritic cells, and by micro-organisms  glycans with lectins in the environment of the inflamed parotid gland tissue could offer (autoreactive) B-cells this alternative selection mechanism.Parotid biopsies were originally collected for diagnostic purposes. Usage of these biopsies for research purposes was obtained on written informed consent with approval from the medical ethical committee of the University Medical Center Groningen, Groningen, the Netherlands.FK, HB, and NB designed the study. HB and ArV recruited the patients and provided clinical data. FS performed the surgical procedures. MD and NV performed/supervised the NGS and the initial screening of the data. AnV performed further analysis of the NGS data and wrote the concept of this manuscript. RB performed the stereotypic RF analysis. NB and FK were involved in writing and discussing the data. All authors critically reviewed the manuscript and approved the final version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


JCR:LA-cp rat model was developed with an autosomal recessive corpulent (cp) trait resulting from a premature stop codon in the extracellular domain of the leptin receptor. Rats that are heterozygous for the cp trait are lean-prone, while those that are homozygous (cp/cp) spontaneously display the pathophysiology of obesity as well as a metabolic syndrome (MetS)-like phenotype. Over the years, there have been formidable scientific contributions that have originated from this rat model, much of which has been reviewed extensively up to 2008. The premise of these earlier studies focused on characterizing the pathophysiology of MetS-like phenotype that was spontaneously apparent in this model. The purpose of this review is to highlight areas of recent advancement made possible by this model including; emerging appreciation of the “thrifty gene” hypothesis in the context of obesity, the concept of how chronic inflammation may drive obesogenesis, the impact of acute forms of inflammation to the brain and periphery during chronic obesity, the role of dysfunctional insulin metabolism on lipid metabolism and vascular damage, and the mechanistic basis for altered vascular function as well as novel parallels between the human condition and the female JCR:LA-cp rat as a model for polycystic ovary disease (PCOS).Obesity and its metabolic complications have emerged as the epidemic of the new millennia. The use of obese rodent models continues to be a productive component of efforts to understand the concomitant metabolic complications of this disease. In 1978, the  Obesity and its metabolic complications have emerged as the epidemic of the new millennia. Fueled by a caloric-dense (and nutrient-poor) food chain that is readily available in most developed countries, the current generation reflects the expression of a human phenotype plagued by the “obesogenic” environment. We have become so efficient at creating foods that target brain reward pathways to stimulate addictiveness and palatability, that we now suffer from the health consequences of not being able to alter our behavior away from this food environment. So too, we are just as guiltily of bestowing this new found talent on the next generation of young adults.JCR:LA-cp rat , has received significant attention. For the last three decades, this model has appeared in the literature every year, often in numerous forms spanning facets of nutrition, endocrinology, metabolic syndrome (MetS), obesity, lipid metabolism, vascular myocardial pathophysiology, and pharmacology  trait], initial breeding stock was sent from National Institutes of Health (NIH) by Dr. Carl T. Hanson to the laboratory of one of the authors (James C. Russell) at the University of Alberta. These rats were the founders of a colony that retained ~3% of the genome derived from the obese spontaneously hypertensive rat (or SHROB). Unlike other NIH colonies, at the time, that were maintained inbred and congenic, the JCR:LA-cp strain has been maintained as a closed outbred colony at the University of Alberta to retain the unknown genetic elements leading to early development of cardiovascular disease (CVD).In 1978, at the fifth backcross to the LA/N strain , a lower production of IFN-γ and decreased IL-1β (LPS) and IL-10 (Con A) was observed , IL-4 (53%), Tnf-γ (31%), and IFN-γ (31%), and twofold greater IL-6 compared to lean-prone animals , including trans-vaccenic acid  in obese-prone animals, further supporting IL-1Ra as a lead candidate for the treatment of ischemic stroke  induced in JCR:LA-cp was used to investigate the effect of infection on vascular and inflammatory responses prior to and after cerebral ischemia  can lead to increased absorption of both lipid and sterol from intestine that can, in turn, exacerbate dyslipidemia and CVD risk. Specifically, we have demonstrated that insulin resistance results in a chronic overproduction and excessive secretion of intestinal-derived apoB48-containing chylomicron particles. This finding is consistent with studies from the JCR:LA-cp rat  may also have independent bioactivity in regulating lipid metabolism. However, few studies have attempted to explore the metabolic pathways that are potentially modulated by VA. Using the obese JCR:LA-cp rat, we have offered a number of contributions to better understand the potential role of VA in activating and thereby regulating lipid metabolism -α and PPAR-γ  have been successful as potent lipid-lowering and insulin-sensitizing therapies for CVD and diabetes-related dyslipidemia , 45. Actfunction . We becafunction , 48. Mor effects , 50. It tabolism , 52. Mos-cp rats , 53. Theble oils .JCR:LA-cp rat, Parnell and Reimer have demonstrated that a 10% dietary blend of prebiotic fibres (inulin and oligofructose) can lower total cholesterol concentrations. The dietary blend is thought stimulate cholesterol excretion in the form of bile as well as reduce hepatic steatosis through a FAS independent mechanism  is an important adaptive process in transient, repetitive coronary artery occlusion, and myocardial ischemia, which occurs in stable farction . In contfarction , 58 and farction . Arteriofarction  and the A-cp rat , 61, 62.A-cp rat –65.JCR:LA-cp obese-prone rat compared to the lean-prone metabolically normal animal, and when miR-145 levels are upregulated in the obese-prone animal, the result is complete restoration of coronary collateral growth -145 and miR-21 are important regulators of arteriogenesis. miR-145 levels are reduced in the l growth . This efl growth . miR-21 l growth  and decrl growth . Conversl growth ; concomil growth . miR-21 JCR:LA-cp rat. The concentration of miR-21 in these animals was also increased in intestinal lymph and in the high-density lipoprotein (HDL) fraction isolated from the lymph ], the reasons for which are multifactorial. We have been very interested in understanding the role that chronic metabolic disease has in exacerbating dyslipidemia and how this mechanistically translates into greater lipid deposition within the arterial wall. Atherogenic cholesterol-dense lipoproteins  are thought to permeate both intact and/or damaged arterial endothelium, become entrapped within the sub-endothelial space, and accumulate, resulting in inflammation and atheroma , 73. AltInterestingly, a series of recent publications authored by the Copenhagen Heart Study group have provided evidence for a major shift in the paradigm of atherogenesis , suggesting that remnant (non-fasting) cholesterol is a major causative factor for ischemic heart disease (IHD) , 80.JCR:LA-cp rat, we have also collected a substantial array of preclinical data showing that remnant lipoproteins carry substantially more cholesterol (due to their size and enrichment) compared to other atherogenic fractions. Using well-established arterial perfusion methodology, we have conducted comparative dual labeling lipoprotein experiments. Cy5-labeled remnants were isolated, purified, and perfused simultaneously with Cy3-labeled LDL . We observed a significantly greater number of LDL particles delivered to the vessel (4.5 ± 1 × 10−9 µg/µm2 tissue) as compared to remnants (0.48 ± 0.15 × 10−9 µg/µm2 tissue) (retention), we observed significantly fewer (55% decrease) LDL particles remaining in the tissue . Additional studies have gone on to show that there is an increased deposition of remnant lipoproteins in arteries from JCR:LA-cp rats as demonstrated by arterial perfusion of equivalent numbers of particles. Mechanistically, we have proposed that this phenomenon may be due to (a) dyslipoproteinemia and/or (b) perturbations in the vessel wall. Crossover perfusion experiments have revealed that increased retention of the number of remnant lipoproteins during insulin resistance was due to differences in arterial vasculature and independent of other factors effecting particle dysfunction per se.Using the  tissue) . Howeverex vivo  is a pharmaceutical compound that selectively reduces intestinal cholesterol absorption by inhibiting the Niemann-pick C1-like 1 (NPC1L1) transporter  while Si ex vivo . It is aJCR:LA-cp rats are consistent with benefits of either EZ + SV therapy on both remnant particle metabolism and corresponding cholesterol  or EZ + SV (−84%) have displayed a significant reduction in the frequency of early stage 2 myocardial lesions or very recent ischemic lesions undergoing scavenging and repair, characteristic for this strain at this age.Ischemic myocardial lesions constitute a critical end point in CVD. Previous studies using the ulinemia . Hearts JCR:LA-cp rat has previously been identified as a model that also includes pathological complications of endothelial dysfunction and myocardial ischemia, in addition to other dysfunctional complications consistent with the MetS . 2-D parasternal long- and short-axis images of the left ventricle (LV) were obtained using a 25-MHz linear-array transducer and doppler probe in anatomical M-mode at the level of papillary muscles at a sweep speed of 150 mm/s. We observed that JCR:LA-cp rats exhibited distinct signs of cardiac dysfunction, and that hearts exhibited a marked increase (~40%) in LV mass vs. their lean counterparts. Echocardiographical analysis also revealed that hearts from JCR:LA-cp rats had an increased early (MV-E) filling velocity (~40%) and reduced late (MV-A) filling (~15%) velocity compared to age-matched lean rats. We further revealed that JCR:LA-cp rats had a restrictive filling pattern, shown by a significantly shortened isovolume relaxation time (IVRT) (~30% decrease). Finally, we found that JCR:LA-cp rats exhibited progressive worsening of diastolic filling properties, with a 1.6-fold increase in the ratio of early to late filling velocity (E/A).The the MetS , 92, 93.function . JCR:LA-JCR:LA-cp rodent model is the only model to spontaneously develop PCOS in conditions of the MetS  has become an increasing public health concern given its association with menstrual dysfunction, infertility, MetS, T2D, and CVD risk , 96. Thethe MetS . The sig-cp rats , 104.JCR:LA-cp rodent model is understanding the distinct mechanisms of androgens and insulin in the cardiometabolic manifestations, particularly dyslipidemia and CVD risk , apoB48, and apoB100  compared to control animals  inhibitor, has confirmed that testosterone action  insulin . Fasting insulin . Interes insulin .In the PCOS-prone model, we have shown that energy restriction and voluntary exercise intervention (4 h/day) at an early life stage can significantly attenuate reproductive and cardiometabolic aberrations . The comThe findings of this work have revealed voluntary exercise has modest effects on cardiometabolic risk factors, and the inclusion of medications that specifically target insulin resistance and dyslipidemia are required to modulate these risk factors in this obese PCOS-prone model. These results have also highlighted the necessity for early intervention with combinations of lifestyle and/or dietary pharmaceutical medication to modulate the hypothalamic–pituitary–ovary axis.JCR:LA-cp rat model has been its suitability to study the progression of metabolic disease from initial stages of overnutrition , resulting in the development of insulin resistance through to the phenotypic complications of hyperinsulinemia and hallmark conditions of the MetS. Many of the advances discussed in this review have made an impact to a better understanding of the human clinical condition. One such example is the revelation of how the intestine integrates and coordinates whole body lipid metabolism more prominently than previously appreciated  and cardiometabolic risk: DV and AD. Impact of JCR:LA-cp rat model to translational outcomes for obesity: SP.Introduction to the clinical problem: SP, WP, and JR. Obesity: testing the “thrifty gene” hypothesis of adaptation to dietary energy intake: AD and WP. Characterization of the altered immune function in obesity using the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.