Multiple In Vitro and In Vivo Regulatory Effects of Budesonide in CD4+ T Lymphocyte Subpopulations of Allergic Asthmatics

Background

Increased activation and increased survival of T lymphocytes characterise bronchial asthma.

Objectives

In this study the effect of budesonide on T cell survival, on inducible co-stimulator T cells (ICOS), on Foxp3 and on IL-10 molecules in T lymphocyte sub-populations was assessed.

Methods

Cell survival (by annexin V binding) and ICOS in total lymphocytes, in CD4+/CD25+ and in CD4+/CD25- and Foxp3 and IL-10 in CD4+/CD25+ and in CD4+/CD25-cells was evaluated, by cytofluorimetric analysis, in mild intermittent asthmatics (n = 19) and in controls (n = 15). Allergen induced T lymphocyte proliferation and the in vivo effects of budesonide in mild persistent asthmatics (n = 6) were also explored.

Results

Foxp3 was reduced in CD4+/CD25- and in CD4+/CD25+ cells and ICOS was reduced in CD4+/CD25+ cells but it was increased in CD4+CD25-in asthmatics when compared to controls. In asthmatics, in vitro, budesonide was able to: 1) increase annexin V binding and to reduce ICOS in total lymphocytes; 2) increase annexin V binding and Foxp3 and to reduce ICOS in CD4+/CD25- cells; 3) reduce annexin V binding and to increase IL-10 and ICOS in CD4+/CD25+ cells; 4) reduce cell allergen induced proliferation. In vivo, budesonide increased ICOS in CD4+/CD25+ while it increased Foxp3 and IL-10 in CD4+/CD25+ and in CD4+/CD25- cells.

Conclusions

Budesonide modulates T cell survival, ICOS, Foxp3 and IL-10 molecules differently in T lymphocyte sub-populations. The findings provided shed light on new mechanisms by which corticosteroids, drugs widely used for the clinical management of bronchial asthma, control T lymphocyte activation.     

High Prevalence of Human Cytomegalovirus Proteins and Nucleic Acids in Primary Breast Cancer and Metastatic Sentinel Lymph Nodes

Background

Breast cancer is a leading cause of death among women worldwide. Increasing evidence implies that human cytomegalovirus (HCMV) infection is associated with several malignancies. We aimed to examine whether HCMV is present in breast cancer and sentinel lymph node (SLN) metastases.

Materials and Methods

Formalin-fixed paraffin-embedded tissue specimens from breast cancer and paired sentinel lymph node (SLN) samples were obtained from patients with (n = 35) and without SLN metastasis (n = 38). HCMV immediate early (IE) and late (LA) proteins were detected using a sensitive immunohistochemistry (IHC) technique and HCMV DNA by real-time PCR.

Results

HCMV IE and LA proteins were abundantly expressed in 100% of breast cancer specimens. In SLN specimens, 94% of samples with metastases (n = 34) were positive for HCMV IE and LA proteins, mostly confined to neoplastic cells while some inflammatory cells were HCMV positive in 60% of lymph nodes without metastases (n = 35). The presence of HCMV DNA was confirmed in 12/12 (100%) of breast cancer and 10/11 (91%) SLN specimens from the metastatic group, but was not detected in 5/5 HCMV-negative, SLN-negative specimens. There was no statistically significant association between HCMV infection grades and progesterone receptor, estrogen receptor alpha and Elston grade status.

Conclusions

The role of HCMV in the pathogenesis of breast cancer is unclear. As HCMV proteins were mainly confined to neoplastic cells in primary breast cancer and SLN samples, our observations raise the question whether HCMV contributes to the tumorigenesis of breast cancer and its metastases.     

Tobacco Smoking Increases Immune Activation and Impairs T-Cell Function in HIV Infected Patients on Antiretrovirals: A Cross-Sectional Pilot Study

Background

The influence of tobacco smoking on the immune system of HIV infected individuals is largely unknown. We investigated the impact of tobacco smoking on immune activation, microbial translocation, immune exhaustion and T-cell function in HIV infected individuals.

Method

HIV infected smokers and non-smokers (n = 25 each) with documented viral suppression on combination antiretroviral therapy and HIV uninfected smokers and non-smokers (n = 15 each) were enrolled. Markers of immune activation (CD38 and HLA-DR) and immune exhaustion (PD1, Tim3 and CTLA4) were analyzed in peripheral blood mononuclear cells (PBMCs) by flow cytometry. Plasma markers of microbial translocation (soluble-CD14 - sCD14 and lipopolysaccharide - LPS) were measured. Antigen specific functions of CD4+ and CD8+ T-cells were measured, by flow cytometry, in PBMCs after 6 hours stimulation with Cytomegalovirus, Epstein-Barr virus and Influenza Virus (CEF) peptide pool.

Results

Compared to non-smokers, smokers of HIV infected and uninfected groups showed significantly higher CD4+ and CD8+ T-cell activation with increased frequencies of CD38+HLA-DR+ cells with a higher magnitude in HIV infected smokers. Expressions of immune exhaustion markers (PD1, Tim3 and CTLA4) either alone or in combinations were significantly higher in smokers, especially on CD4+ T-cells. Compared to HIV uninfected non-smokers, microbial translocation (sCD14 and LPS) was higher in smokers of both groups and directly correlated with CD4+ and CD8+ T-cell activation. Antigen specific T-cell function showed significantly lower cytokine response of CD4+ and CD8+ T-cells to CEF peptide-pool stimulation in smokers of both HIV infected and uninfected groups.

Conclusions

Our results suggest that smoking and HIV infection independently influence T-cell immune activation and function and together they present the worst immune profile. Since smoking is widespread among HIV infected individuals, studies are warranted to further evaluate the cumulative effect of smoking on impairment of the immune system and accelerated disease progression.     

Frequency of Chromosomally-Integrated Human Herpesvirus 6 in Children with Acute Lymphoblastic Leukemia

Introduction

Human herpesvirus 6 (HHV-6) is a ubiquitous pathogen infecting nearly 100% of the human population. Of these individuals, between 0.2% and 1% of them carry chromosomally-integrated HHV-6 (ciHHV-6). The biological consequences of chromosomal integration by HHV-6 remain unknown.

Objective

To determine and compare the frequency of ciHHV-6 in children with acute lymphoblastic leukemia to healthy blood donors.

Methodology

A total of 293 DNA samples from children with pre-B (n = 255), pre-pre-B (n = 4), pre-T (n = 26) and undetermined (n = 8) leukemia were analyzed for ciHHV-6 by quantitative TaqMan PCR (QPCR) using HHV-6 specific primers and probe. As control, DNA samples from 288 healthy individuals were used. Primers and probe specific to the cellular GAPDH gene were used to estimate integrity and DNA content.

Results

Out of 293 DNA samples from the leukemic cohort, 287 contained amplifiable DNA. Of these, only 1 (0.35%) contained ciHHV-6. Variant typing indicates that the ci-HHV-6 corresponds to variant A. None of the 288 DNA samples from healthy individuals contained ciHHV-6.

Conclusion

The frequency of ciHHV-6 in children with acute lymphoblastic leukemia is similar (p = 0.5) to that of healthy individuals. These results suggest that acute lymphoblastic leukemia does not originate as a consequence to integration of HHV-6 within the chromosomes.     

Detection of Circulating Tumor Cell Subpopulations in Patients with Head and Neck Squamous Cell Carcinoma (HNSCC)

Background

Since image based diagnostic tools fail to detect early metastasis in head and neck squamous cell carcinoma (HNSCC) it is crucial to develop minimal invasive diagnostic methods. A promising approach is to identify and characterize circulating tumor cells (CTC) in the peripheral blood of HNSCC patients. In this pilot study, we assessed which non-hematopoietic cell types are identifiable and whether their numbers differ in pre- and postoperative blood samples.

Methods

20 ml citrated peripheral blood was taken from 10 HNSCC patients before and after curative resection. CTC were enriched using density gradient centrifugation. CTC presence was verified by multi-immunofluorescence staining against cytokeratin (CK; epithelial), N-cadherin (mesenchymal); CD133 (stem-cell), CD45 (hematopoietic) and DAPI (nucleus). Individual cell type profiles were analyzed.

Results

We were able to detect cells with epithelial properties like CK+/N-cadherin−/CD45− and CK+/CD133−/CD45− as well as cells with mesenchymal features such as N-cadherin+/CK−/CD45− and cells with both characteristics like N-cadherin+/CK+/CD45−. We also observed cells showing stem cell-like features like CD133+/CK−/CD45− and cells with both epithelial and stem cell-like features such as CD133+/CK+/CD45−. The number of CK positive cells (p = 0.002), N-cadherin positive cells (p = 0.002) and CD133 positive cells (p = 0.01) decreased significantly after resection. Kaplan-Meier test showed that the survival was significantly shorter when N-cadherin+ cells were present after resection (p = 0.04; 474 vs. 235 days; [HR] = 3.1).

Conclusions

This is - to the best of our knowledge- the first pilot study identifying different CTC populations in peripheral blood of HNSCC patients and showing that these individual cell type profiles may have distinct clinical implications.     

Notch Signaling in Descending Thoracic Aortic Aneurysm and Dissection

Background

Descending thoracic aortic aneurysm and dissection (DTAAD) is characterized by progressive medial degeneration, which may result from excessive tissue destruction and insufficient repair. Resistance to tissue destruction and aortic self-repair are critical in preventing medial degeneration. The signaling pathways that control these processes in DTAAD are poorly understood. Because Notch signaling is a critical pathway for cell survival, proliferation, and tissue repair, we examined its activation in DTAAD.

Methods

We studied descending thoracic aortic tissue from patients with sporadic thoracic aortic aneurysm (TAA; n = 14) or chronic thoracic aortic dissection (TAD; n = 16) and from age-matched organ donors (n = 12). Using western blot, real-time RT-PCR, and immunofluorescence staining, we examined aortic tissue samples for the Notch ligands Delta-like 1, Delta-like 4 (DLL1/4), and Jagged1; the Notch receptor 1 (Notch1); the Notch1 intracellular domain (NICD); and Hes1, a downstream target of Notch signaling.

Results

Western blots and RT-PCR showed higher levels of the Notch1 protein and mRNA and the NICD and Hes1 proteins in both TAA and TAD tissues than in control tissue. However, immunofluorescence staining showed a complex pattern of Notch signaling in the diseased tissue. The ligand DLL1/4 and Notch1 were significantly decreased and NICD and Hes1 were rarely detected in medial vascular smooth muscle cells (VSMCs) in both TAA and TAD tissues, indicating downregulation of Notch signaling in aortic VSMCs. Interestingly Jagged1, NICD, and Hes1 were highly present in CD34+ stem cells and Stro-1+ stem cells in aortas from TAA and TAD patients. NICD and Hes1 were also detected in most fibroblasts and macrophages that accumulated in the aortic wall of DTAAD patients.

Conclusions

Notch signaling exhibits a complex pattern in DTAAD. The Notch pathway is impaired in medial VSMCs but activated in stem cells, fibroblasts, and macrophages.     

Validation of the Cardiosphere Method to Culture Cardiac Progenitor Cells from Myocardial Tissue

Background

At least four laboratories have shown that endogenous cardiac progenitor cells (CPCs) can be grown directly from adult heart tissue in primary culture, as cardiospheres or their progeny (cardiosphere-derived cells, CDCs). Indeed, CDCs are already being tested in a clinical trial for cardiac regeneration. Nevertheless, the validity of the cardiosphere strategy to generate CPCs has been called into question by reports based on variant methods. In those reports, cardiospheres are argued to be cardiomyogenic only because of retained cardiomyocytes, and stem cell activity has been proposed to reflect hematological contamination. We use a variety of approaches (including genetic lineage tracing) to show that neither artifact is applicable to cardiospheres and CDCs grown using established methods, and we further document the stem cell characteristics (namely, clonogenicity and multilineage potential) of CDCs.

Methodology/Principal Findings

CPCs were expanded from human endomyocardial biopsies (n = 160), adult bi-transgenic MerCreMer-Z/EG mice (n = 6), adult C57BL/6 mice (n = 18), adult GFP⁺ C57BL/6 transgenic mice (n = 3), Yucatan mini pigs (n = 67), adult SCID beige mice (n = 8), and adult Wistar-Kyoto rats (n = 80). Cellular yield was enhanced by collagenase digestion and process standardization; yield was reduced in altered media and in specific animal strains. Heparinization/retrograde organ perfusion did not alter the ability to generate outgrowth from myocardial sample. The initial outgrowth from myocardial samples was enriched for sub-populations of CPCs (c-Kit⁺), endothelial cells (CD31⁺, CD34⁺), and mesenchymal cells (CD90⁺). Lineage tracing using MerCreMer-Z/EG transgenic mice revealed that the presence of cardiomyocytes in the cellular outgrowth is not required for the generation of CPCs. Rat CDCs are shown to be clonogenic, and cloned CDCs exhibit spontaneous multineage potential.

Conclusions/Significance

This study demonstrates that direct culture and expansion of CPCs from myocardial tissue is simple, straightforward, and reproducible when appropriate techniques are used.     

Sequential Anti-Cytomegalovirus Response Monitoring May Allow Prediction of Cytomegalovirus Reactivation after Allogeneic Stem Cell Transplantation

Background

Reconstitution of cytomegalovirus-specific CD3⁺CD8⁺ T cells (CMV-CTLs) after allogeneic hematopoietic stem cell transplantation (HSCT) is necessary to bring cytomegalovirus (CMV) reactivation under control. However, the parameters determining protective CMV-CTL reconstitution remain unclear to date.

Design and Methods

In a prospective tri-center study, CMV-CTL reconstitution was analyzed in the peripheral blood from 278 patients during the year following HSCT using 7 commercially available tetrameric HLA-CMV epitope complexes. All patients included could be monitored with at least CMV-specific tetramer.

Results

CMV-CTL reconstitution was detected in 198 patients (71%) after allogeneic HSCT. Most importantly, reconstitution with 1 CMV-CTL per µl blood between day +50 and day +75 post-HSCT discriminated between patients with and without CMV reactivation in the R+/D+ patient group, independent of the CMV-epitope recognized. In addition, CMV-CTLs expanded more daramtaically in patients experiencing only one CMV-reactivation than those without or those with multiple CMV reactivations. Monitoring using at least 2 tetramers was possible in 63% (n = 176) of the patients. The combinations of particular HLA molecules influenced the numbers of CMV-CTLs detected. The highest CMV-CTL count obtained for an individual tetramer also changed over time in 11% of these patients (n = 19) resulting in higher levels of HLA-B*0801 (IE-1) recognizing CMV-CTLs in 14 patients.

Conclusions

Our results indicate that 1 CMV-CTL per µl blood between day +50 to +75 marks the beginning of an immune response against CMV in the R+/D+ group. Detection of CMV-CTL expansion thereafter indicates successful resolution of the CMV reactivation. Thus, sequential monitoring of CMV-CTL reconstitution can be used to predict patients at risk for recurrent CMV reactivation.     

T-Cell Phenotypes,Apoptosis and Inflammation in HIV+ Patients on Virologically Effective cART with Early Atherosclerosis

Objective

We investigated the potential relationship between T-cell phenotype, inflammation, endotoxemia, and atherosclerosis evaluated by carotid intima-media thickness (IMT) in a cohort of HIV-positive patients undergoing long-term virologically suppressive combination antiretroviral therapy (cART).

Design

We studied 163 patients receiving virologically suppressive cART.

Methods

We measured IMT (carotid ultrasound); CD4+/CD8+ T-cell activation (CD38, CD45R0), differentiation (CD127), apoptosis (CD95), and senescence (CD28, CD57) (flow cytometry); plasma sCD14, IL-6, TNF- α, sVCAM-1, hs-CRP, anti-CMV IgG (ELISA); LPS (LAL). The results were compared by Mann-Whitney, Kruskal-Wallis or Chi-square tests, and factors associated with IMT were evaluated by multivariable logistic regression.

Results

Of 163 patients, 112 demonstrated normal IMT (nIMT), whereas 51 (31.3%) had pathological IMT (pIMT: ≥1 mm). Of the patients with pIMT, 22 demonstrated an increased IMT (iIMT), and 29 were shown to have plaques. These patient groups had comparable nadir and current CD4+, VLs and total length of time on cART. Despite similar proportions of CD38-expressing CD8+ cells (p = .95), pIMT patients exhibited higher activated memory CD8+CD38+CD45R0+ cells (p = .038) and apoptotic CD4+CD95+ (p = .01) and CD8+CD95+ cells (p = .003). In comparison to nIMT patients, iIMT patients tended to have lower numbers of early differentiated CD28+CD57− memory CD4+ (p = .048) and CD28–CD57−CD8+ cells (p = .006), both of which are associated with a higher proliferative potential. Despite no differences in plasma LPS levels, pIMT patients showed significantly higher circulating levels of sCD14 than did nIMT patients (p = .046). No differences in anti-CMV IgG was shown. Although circulating levels of sCD14 seemed to be associated with a risk of ATS in an unadjusted analysis, this effect was lost after adjusting for classical cardiovascular predictors.

Conclusions

Despite the provision of full viral suppression by cART, a hyperactivated, pro-apoptotic T-cell profile characterizes HIV-infected patients with early vascular damage, for whom the potential contribution of subclinical endotoxemia and anti-CMV immunity should be investigated further.     

Multiparameter Phospho-Flow Analysis of Lymphocytes in Early Rheumatoid Arthritis: Implications for Diagnosis and Monitoring Drug Therapy

Background

The precise mechanisms involved in the initiation and progression of rheumatoid arthritis (RA) are not known. Early stages of RA often have non-specific symptoms, delaying diagnosis and therapy. Additionally, there are currently no established means to predict clinical responsiveness to therapy. Immune cell activation is a critical component therefore we examined the cellular activation of peripheral blood mononuclear cells (PBMCs) in the early stages of RA, in order to develop a novel diagnostic modality.

Methods and Findings

PBMCs were isolated from individuals diagnosed with early RA (ERA) (n = 38), longstanding RA (n = 10), osteoarthritis (OA) (n = 19) and from healthy individuals (n = 10). PBMCs were examined for activation of 15 signaling effectors, using phosphorylation status as a measure of activation in immunophenotyped cells, by flow cytometry (phospho-flow). CD3+CD4+, CD3+CD8+ and CD20+ cells isolated from patients with ERA, RA and OA exhibited activation of multiple phospho-epitopes. ERA patient PBMCs showed a bias towards phosphorylation-activation in the CD4+ and CD20+ compartments compared to OA PBMCs, where phospho-activation was primarily observed in CD8+ cells. The ratio of phospho (p)-AKT/p-p38 was significantly elevated in patients with ERA and may have diagnostic potential. The mean fluorescent intensity (MFI) levels for p-AKT and p-H3 in CD4+, CD8+ and CD20+ T cells correlated directly with physician global assessment scores (MDGA) and DAS (disease activity score). Stratification by medications revealed that patients receiving leflunomide, systemic steroids or anti-TNF therapy had significant reductions in phospho-specific activation compared with patients not receiving these therapies. Correlative trends between medication-associated reductions in the levels of phosphorylation of specific signaling effectors and lower disease activity were observed.

Conclusions

Phospho-flow analysis identified phosphorylation-activation of specific signaling effectors in the PB from patients with ERA. Notably, phosphorylation of these signaling effectors did not distinguish ERA from late RA, suggesting that the activation status of discrete cell populations is already established early in disease. However, when the ratio of MFI values for p-AKT and p-p38 is >1.5, there is a high likelihood of having a diagnosis of RA. Our results suggest that longitudinal sampling of patients undergoing therapy may result in phospho-signatures that are predictive of drug responsiveness.     

Discordance in CD4+T-Cell Levels and Viral Loads with Co-Occurrence of Elevated Peripheral TNF-α and IL-4 in Newly Diagnosed HIV-TB Co-Infected Cases

Background

Cytokines are the hallmark of immune response to different pathogens and often dictate the disease outcome. HIV infection and tuberculosis (TB) are more destructive when confronted together than either alone. Clinical data related to the immune status of HIV-TB patients before the initiation of any drug therapy is not well documented. This study aimed to collect the baseline information pertaining to the immune status of HIV-TB co-infected patients and correlate the same with CD4+T cell levels and viral loads at the time of diagnosis prior to any drug therapy.

Methodology/Principal Findings

We analyzed the cytokines, CD4+T cell levels and viral loads to determine the immune environment in HIV-TB co-infection. The study involved four categories namely, Healthy controls (n = 57), TB infected (n = 57), HIV infected (n = 59) and HIV-TB co-infected (n = 57) patients. The multi-partite comparison and correlation between cytokines, CD4+T-cell levels and viral loads prior to drug therapy, showed an altered TH1 and TH2 response, as indicated by the cytokine profiles and skewed IFN-γ/IL-10 ratio. Inadequate CD4+T cell counts in HIV-TB patients did not correlate with high viral loads and vice-versa. When compared to HIV category, 34% of HIV-TB patients had concurrent high plasma levels of IL-4 and TNF-α at the time of diagnosis. TB relapse was observed in 5 of these HIV-TB co-infected patients who also displayed high IFN-γ/IL-10 ratio.

Conclusion/Significance

With these studies, we infer (i) CD4+T-cell levels as baseline criteria to report the disease progression in terms of viral load in HIV-TB co-infected patients can be misleading and (ii) co-occurrence of high TNF-α and IL-4 levels along with a high ratio of IFN-γ/IL-10, prior to drug therapy, may increase the susceptibility of HIV-TB co-infected patients to hyper-inflammation and TB relapse.     

Absence of CD34 on Murine Skeletal Muscle Satellite Cells Marks a Reversible State of Activation during Acute Injury

Background

Skeletal muscle satellite cells are myogenic progenitors that reside on myofiber surface beneath the basal lamina. In recent years satellite cells have been identified and isolated based on their expression of CD34, a sialomucin surface receptor traditionally used as a marker of hematopoietic stem cells. Interestingly, a minority of satellite cells lacking CD34 has been described.

Methodology/Principal Findings

In order to elucidate the relationship between CD34+ and CD34- satellite cells we utilized fluorescence-activated cell sorting (FACS) to isolate each population for molecular analysis, culture and transplantation studies. Here we show that unless used in combination with α7 integrin, CD34 alone is inadequate for purifying satellite cells. Furthermore, the absence of CD34 marks a reversible state of activation dependent on muscle injury.

Conclusions/Significance

Following acute injury CD34- cells become the major myogenic population whereas the percentage of CD34+ cells remains constant. In turn activated CD34- cells can reverse their activation to maintain the pool of CD34+ reserve cells. Such activation switching and maintenance of reserve pool suggests the satellite cell compartment is tightly regulated during muscle regeneration.     

Helminth-Associated Systemic Immune Activation and HIV Co-receptor Expression: Response to Albendazole/Praziquantel Treatment

Background

It has been hypothesized that helminth infections increase HIV susceptibility by enhancing systemic immune activation and hence contribute to elevated HIV-1 transmission in sub-Saharan Africa.

Objective

To study systemic immune activation and HIV-1 co-receptor expression in relation to different helminth infections and in response to helminth treatment.

Methods

HIV-negative adults with (n = 189) or without (n = 57) different helminth infections, as diagnosed by Kato-Katz, were enrolled in Mbeya, Tanzania. Blinded to helminth infection status, T cell differentiation (CD45RO, CD27), activation (HLA-DR, CD38) and CCR5 expression was determined at baseline and 3 months after Albendazole/Praziquantel treatment. Plasma cytokine levels were compared using a cytometric bead array.

Results

Trichuris and Ascaris infections were linked to increased frequencies of “activated” CD4 and/or CD8 T cells (p<0.05), whereas Hookworm infection was associated with a trend towards decreased HLA-DR⁺ CD8 T cell frequencies (p = 0.222). In Trichuris infected subjects, there was a linear correlation between HLA-DR⁺ CD4 T cell frequencies and the cytokines IL-1β and IL-10 (p<0.05). Helminth treatment with Albendazole and Praziquantel significantly decreased eosinophilia for S. mansoni and Hookworm infections (p<0.005) but not for Trichuris infection and only moderately modulated T cell activation. CCR5 surface density on memory CD4 T cells was increased by 1.2-fold during Trichuris infection (p-value: 0.053) and reduced after treatment (p = 0.003).

Conclusions

Increased expression of T cell activation markers was associated with Trichuris and Ascaris infections with relatively little effect of helminth treatment.     

Cranialization of the Frontal Sinus for Secondary Mucocele Prevention following Open Surgery for Benign Frontal Lesions

Objective

To compare frontal sinus cranialization to obliteration for future prevention of secondary mucocele formation following open surgery for benign lesions of the frontal sinus.

Study Design

Retrospective case series.

Setting

Tertiary academic medical center.

Patients

Sixty-nine patients operated for benign frontal sinus pathology between 1994 and 2011.

Interventions

Open excision of benign frontal sinus pathology followed by either frontal obliteration (n = 41, 59%) or frontal cranialization (n = 28, 41%).

Main Outcome Measures

The prevalence of post-surgical complications and secondary mucocele formation were compiled.

Results

Pathologies included osteoma (n = 34, 49%), mucocele (n = 27, 39%), fibrous dysplasia (n = 6, 9%), and encephalocele (n = 2, 3%). Complications included skin infections (n = 6), postoperative cutaneous fistula (n = 1), telecanthus (n = 4), diplopia (n = 3), nasal deformity (n = 2) and epiphora (n = 1). None of the patients suffered from postoperative CSF leak, meningitis or pneumocephalus. Six patients, all of whom had previously undergone frontal sinus obliteration, required revision surgery due to secondary mucocele formation. Statistical analysis using non-inferiority test reveal that cranialization of the frontal sinus is non-inferior to obliteration for preventing secondary mucocele formation (P<0.0001).

Conclusion

Cranialization of the frontal sinus appears to be a good option for prevention of secondary mucocele development after open excision of benign frontal sinus lesions.     

Identification of Biological Properties of Intralymphatic Tumor Related to the Development of Lymph Node Metastasis in Lung Adenocarcinoma

Background

Intralymphatic tumors in the extratumoral area are considered to represent the preceding phase of lymph node metastasis. The aim of this study was to clarify the biological properties of intralymphatic tumors susceptible to the development of lymph node metastasis, with special reference to the expression of cancer initiating/stem cell (CIC/CSC) related markers in cancer cells and the number of infiltrating stromal cells.

Material and Methods

Primary lung adenocarcinomas with lymphatic permeation in the extratumoral area were retrospectively examined (n = 107). We examined the expression levels of CIC/CSC related markers including ALDH1, OCT4, NANOG, SOX2 and Caveolin-1 in the intralymphatic cancer cells to evaluate their relationship to lymph node metastasis. Moreover, the number of infiltrating stromal cells expressing CD34, α-smooth muscle actin, and CD204 were also evaluated.

Results

Among the intralymphatic tissues, low ALDH1 expression in cancer cells, high SOX2 expression in cancer cells, and a high number of CD204(+) macrophages were independent predictive factors for lymph node metastasis (P = 0.004, P = 0.008, and P = 0.028, respectively). Among these factors, only low ALDH1 expression in cancer cells was significantly correlated with the farther spreading of lymph node metastasis (mediastinal lymph node, pathological N2) (P = 0.046) and the metastatic lymph node ratio (metastatic/resected) (P = 0.028). On the other hand, in the primary tumors, ALDH1 expression in the cancer cells was not associated with lymph node metastasis. Intralymphatic cancer cells expressing low ALDH1 levels exhibited lower E-cadherin expression levels than cancer cells with high levels of ALDH1 expression (P = 0.015).

Conclusions

Intralymphatic cancer cells expressing low levels of ALDH1 and infiltrating macrophages expressing CD204 have a critical impact on lymph node metastasis. Our study also highlighted the significance of evaluating the biological properties of intralymphatic tumors for tumor metastasis.     

Decreased NK Cell FcRγ in HIV-1 Infected Individuals Receiving Combination Antiretroviral Therapy: a Cross Sectional Study

Background

FcRγ is an immunoreceptor tyrosine-based activation motif (ITAM)-signalling protein essential for immunoreceptor signaling and monocyte, macrophage and NK cell function. Previous study from our laboratory showed that FcRγ is down-regulated in HIV-infected macrophages in vitro. FcRγ expression in immune cells present in HIV-infected individuals is unknown.

Methodology/Principal Findings

We compared FcRγ expression in peripheral blood mononuclear cells isolated from HIV-1-infected individuals receiving combination antiretroviral therapy and healthy, HIV-1-uninfected individuals. FcRγ mRNA and protein levels were measured using quantitative real-time PCR and immunoblotting, respectively. CD56⁺ CD94⁺ lymphocytes isolated from blood of HIV-1 infected individuals had reduced FcRγ protein expression compared to HIV-uninfected individuals (decrease = 76.8%, n = 18 and n = 12 respectively, p = 0.0036). In a second group of patients, highly purified NK cells had reduced FcRγ protein expression compared to uninfected controls (decrease = 50.2%, n = 9 and n = 8 respectively, p = 0.021). Decreased FcRγ expression in CD56+CD94+ lymphocytes was associated with reduced mRNA (51.7%, p = 0.021) but this was not observed for the smaller group of patients analysed for NK cell expression (p = 0.36).

Conclusion/Significance

These data suggest biochemical defects in ITAM-dependent signalling within NK cells in HIV-infected individuals which is present in the context of treatment with combination antiretroviral therapy.     

Ex vivo expanded human cord blood-derived hematopoietic progenitor cells induce lung growth and alveolarization in injured newborn lungs

Background

We investigated the capacity of expanded cord blood-derived CD34+ hematopoietic progenitor cells to undergo respiratory epithelial differentiation ex vivo, and to engraft and attenuate alveolar disruption in injured newborn murine lungs in vivo.

Methods

Respiratory epithelial differentiation was studied in CD34+ cells expanded in the presence of growth factors and cytokines (“basic” medium), in one group supplemented with dexamethasone (“DEX”). Expanded or freshly isolated CD34+ cells were inoculated intranasally in newborn mice with apoptosis-induced lung injury. Pulmonary engraftment, lung growth and alveolarization were studied at 8 weeks post-inoculation.

Results

SP-C mRNA expression was seen in 2/7 CD34+ cell isolates expanded in basic media and in 6/7 isolates expanded in DEX, associated with cytoplasmic SP-C immunoreactivity and ultrastructural features suggestive of type II cell-like differentiation. Administration of expanding CD34+ cells was associated with increased lung growth and, in animals treated with DEX-exposed cells, enhanced alveolar septation. Freshly isolated CD34+ cells had no effect of lung growth or remodeling. Lungs of animals treated with expanded CD34+ cells contained intraalveolar aggregates of replicating alu-FISH-positive mononuclear cells, whereas epithelial engraftment was extremely rare.

Conclusion

Expanded cord blood CD34+ cells can induce lung growth and alveolarization in injured newborn lungs. These growth-promoting effects may be linked to paracrine or immunomodulatory effects of persistent cord blood-derived mononuclear cells, as expanded cells showed limited respiratory epithelial transdifferentiation.