Glenda Ernst

Neutrophils suppress γδ T-cell function

γδ T cells have been shown to stimulate the recruitment and activation of neutrophils through the release of a range of cytokines and chemokines. Here, we investigated the reverse relationship, showing that human neutrophils suppress the function of human blood γδ T cells. We show that the upregulation of CD25 and CD69 expression, the production of IFN‐γ, and the proliferation of γδ T cells induced by (E)‐1‐hydroxy‐2‐methylbut‐2‐enyl 4‐diphosphate are inhibited by neutrophils. Spontaneous activation of γδ T cells in culture is also suppressed by neutrophils. We show that inhibitors of prostaglandin E2 and arginase I do not exert any effect, although, in contrast, catalase prevents the suppression of γδ T cells induced by neutrophils, suggesting the participation of neutrophil‐derived ROS. We also show that the ROS‐generating system xanthine/xanthine oxidase suppresses γδ T cells in a similar fashion to neutrophils, while neutrophils from chronic granulomatous disease patients only weakly inhibit γδ T cells. Our results reveal a bi‐directional cross‐talk between γδ T cells and neutrophils: while γδ T cells promote the recruitment and the activation of neutrophils to fight invading pathogens, neutrophils in turn suppress the activation of γδ T cells to contribute to the resolution of inflammation.

Interaction of CD44 with Different Forms of Hyaluronic Acid. Its Role in Adhesion and Migration of Tumor Cells

Interaction between hyaluronic acid (HA) and CD44 has been considered a key event in tumor invasion and metastasis. HA is a linear, high molecular weight glycosaminoglycan in its native state, but fragmented low molecular forms are found at sites of neoplastic or inflammatory infiltrates. Both high and low molecular weights HA are involved in diverse biological functions. In this study, we used two clonal variants of a T cell murine lymphoma designated LBLa and LBLc. These cell lines were found to differ in their in vivo and in vitro growth rates. LBLa grew faster and exhibited an enhanced invasive capacity as compared to LBLc. In contrast, cell lines did not differ in the expression of surface markers (CD8, CD24, CD25, CD44, and CD18), or in their capacity to bind HA. However, LBLa cells exhibited higher capacity to migrate to low molecular weight HA than did LBLc. Migration was mediated by CD44 since it was abrogated by anti-CD44 monoclonal antibody as well as by hyaluronidase. We suggest that interaction between CD44 and low molecular weight HA may trigger migration mechanisms in LBLa cells, thus contributing to enhanced invasive cell capacity.

Combined chemotherapy and ALA-based photodynamic therapy in leukemic murine cells

The effects of combined administration of doxorubicin (DOX) and vincristine (VCR), with 5-aminolevulinic acid photodynamic treatment (ALA-PDT), were analyzed in sensitive murine leukemic cell lines (LBR-) and DOX and VCR chemoresistant LBR-D160 and LBR-V160 cell lines. Low doses of DOX and VCR increased anti-cancer effect of ALA-PDT in LBR-cells. Decrease in cell survival was higher when the combination VCR+ALA-PDT was used compared to DOX+ALA-PDT. Resistant cell lines LBR-D160 and LBR-V160 were sensitive to ALA-PDT; however, no changes occured when combining therapies. Thus, ALA-PDT can overcome drug resistance and is a good candidate for using treating multidrug resistant (MDR) cells.

Role of EMSE and STESS scores in the outcome evaluation of status epilepticus

Status epilepticus (SE) is a severe neurological condition with significant morbidity and mortality. A reliable tool for prognosis is needed to take decision regarding treatment strategies. We compared 2 available prognostic scores of outcome: the Status Epilepticus Severity Score (STESS) and the Epidemiology-based Mortality score in SE (EMSE). We included 46 patients with SE evaluated out the last 5years in our hospital. We excluded patients with postanoxic encephalopathy or incomplete data. Among the 46 patients with SE, in-hospital mortality was 28%. The receiver operating characteristic (ROC) curve for predicting of death by STESS had an area under the curve (AUC) of 0.80 with cutoff point ≥4. The best EMSE variable combination to predict mortality was EMSE-AEL using an optimized cutoff point of 34 (age/etiology/loss of consciousness) with an area under the ROC of 0.79. The STESS and EMSE would be useful tools to predict in-hospital mortality in SE.

Coelomocyte locomotion in the sipunculan Themiste petricola induced by exogenous and endogenous chemoattractants: role of a CD44-like antigen–HA interaction

Cell migration is a key event in the invertebrate immuno-defense system. Microbial products like lipopolysacharide (LPS) and formyl-methyl-leucyl-phenylalanine (fMLP) promote cell recruitment to sites of infection. In mammals, complement activation by factors such as zymosan induces C5a production, which influences leukocyte migration. The endogenous factor hyaluronic acid (HA), an extracellular matrix component, also promotes cell migration through its receptor CD44. We evaluated whether coelomocytes from the sipunculan worm T. petricola migrated towards LPS, fMLP, or zymosan treated plasma (ZTP) and if HA was involved in coelomocyte migration and adhesion. We also evaluated if antibodies specific for mouse HA receptor CD44 inhibited any of the effects induced by HA. Using microchemotaxis chambers we found that coelomocytes migrated towards exogenously and endogenously derived chemoattractants. We also observed that HA was a potent chemotactic signal and that coelomocytes adhered strongly to plates coated with LMW-HA but not with HMW-HA. In addition we found that these HA mediated effects were blocked by the monoclonal antibody IM7 directed to mouse CD44, suggesting that a CD44-like cross-reactive antigen might play a role in HA mediated coelomocyte locomotion.

Dissimilar invasive and metastatic behavior of vincristine and doxorubicin-resistant cell lines derived from a murine T cell lymphoid leukemia

Multidrug resistance (MDR) lines from a murine T-cell lymphoid leukemia were selected in increasing vincristine (VCR) or doxorubicin (DOX) concentrations. Surface markers were determined by flow cytometry in both resistant (LBR-V160 and LBR-D160) and sensitive (LBR-) cell lines. Results obtained revealed similar expression of CD25, CD24, CD8, CD4, C18 and CD44, while differences in binding to hyaluronic acid (HA) were found. LBR- and LBR-D160 bound to HA only after phorbol ester (PMA) activation, while LBR-V160 failed to bind HA even after PMA treatment. Histopathological analysis disclosed that LBR-V160 was less invasive than LBR- and LBR-D160 cell lines. In vitro growth of cell lines analyzed by sulforhodamine-B uptake showed that doubling time for the three lines was 10.24 h (LBR-), 16.75 h (LBR-V160) and 16.29 h (LBR-D160). Mortality rate was determined after i.p. injection of 104 cells. Mice inoculated with LBR- died at 23 (± 2.11) days, while those inoculated with LBR-V160 or LBR-D160 died at 41 (± 9.53) or 41 (± 4.96) days, respectively. Our results demonstrated that leukemic murine T cells cultured in the long-term presence of VCR or DOX not only presented changes in the resistance phenotype but also variations in their growth and metastatic pattern.

Increased hyaluronan levels and decreased dendritic cell activation are associated with tumor invasion in murine lymphoma cell lines.

Hyaluronan (HA), a component of the extracellular matrix surrounding tumors, modulates tumor progression and the immune response. Dendritic cells (DC) may tolerize or stimulate immunity against cancer. In this report, we study the association between tumor progression, HA levels and DC activation in a lymphoma model. Mice injected with the cells with highest invasive capacity (LBR-) presented increased HA in serum and lymph nodes, and decreased DC activation in infiltrated lymph nodes and liver. These findings could be related to lack of an effective antitumor immune response and suggest that serum HA levels could have a prognostic value in hematological malignancies.

Chromosome studies of murine T-cell lymphoid leukemia and derived cell lines.

Several cell lines were previously established from a spontaneous murine T-cell leukemia (LB). The aim of this study was to analyze the G- and C-banded karyotypes of the parental LB tumor cells and the derived cell lines. A sensitive cell line (LBL) from which two sublines originated, as well as Vincristine (LBR-V160) and Doxorubicin (LBR-D160) resistant cell lines, were used. Our results showed that LB cells had a pseudo-diploid karyotype with 40 acrocentric chromosomes in which trisomy of chromosome 14 was the most relevant alteration. The sensitive cell line showed this alteration in all metaphases studied; no changes in karyotypes were observed in either subline, despite their dissimilar morphology and growth patterns. In contrast, both resistant lines displayed a more heterogeneous karyotype with no common markers, except for the finding that chromosome 5 was involved in a trisomy in LBR-V160 and in a translocation with chromosome 12 in LBR-D160. Taking into account that the mdr genes are located in chromosome 5, these results suggest a possible association between such alterations and the acquisition of drug resistance.

“Diagnosis of sleep apnea in network” respiratory polygraphy as a decentralization strategy

Introduction Obstructive sleep apnea syndrome (OSA) is diagnosed through polysomnography (PSG) or respiratory polygraphy (RP). Self-administered home-based RP using devices with data transmission could facilitate diagnosis in distant populations. The purpose of this work was to describe a telemedicine initiative using RP in four satellite outpatient care clinics (OCC) of Buenos Aires Hospital Británico Central (HBC). Materials and methods OCC technicians were trained both in the use of RP. Raw signals were sent to HBC via intranet software for scoring and final report. Results During a 24-month 499 RP were performed in 499 patients: 303 men (60.7%) with the following characteristics (mean and standard deviation): valid time for manual analysis: 392.8 min (±100.1), AHI: 17.05 (±16.49 and percentile 25–75 [Pt]: 5–23) ev/hour, ODI (criterion 3%): 18.05 (±16.48 and Pt 25–75: 6–25) ev/hour, and time below 90% (T<90): 17.9% (±23.4 and Pt 25–75: 1–23). The distribution of diagnoses (absolute value and percentage) was: normal (66/13%), snoring (70/14%), mild (167/33.5%), moderate (110/22%), and severe (86/17.2%). Continuous positive airway pressure (CPAP) was indicated for 191 patients (38.6%). Twenty recordings (4%) were considered invalid and the RP had to be repeated. PSG at HBC was indicated in 60 (12.1%) cases (mild OSA or normal AHI with high ESS or cardiovascular disease). Conclusions Physicians were able to diagnosis OSA by doing portable respiratory polygraphy at distance. The remote diagnosis strategy presented short delays, safe data transmission, and low rate of missing data.

Simulated intention-to-treat analysis based on clinical parameters ofpatients at high risk for sleep apnea derivated to respiratorypolygraphy

Purpose Obstructive Sleep Apnea-Hypopnea Syndrome (OSAS) is a public health problem. We designed a pilot study to validate empiric indication of CPAP therapy in a population with moderate-to-high pre-test probabilities who underwent self-administered home-based respiratory polygraphy (RP). Methods A cross-sectional simulation study was performed. CPAP therapy could be indicated by two independent blind observers. Observer 1´s decision was based on the results of STOP-BANG (SBQ) and Epworth Sleepiness Scale (ESS) and Observer 2 used all objective data provided by RP + SBQ + ESS. Results We evaluated 1763 patients; 1060 men and 703 women (39.2%) with a mean age of 53.6±13.8 and a body mass index (BMI) of 32.8±7.5 kg/m2. We found evidence of mild (34.1%), moderate (26.6%), and severe (18.3%) There were Apnea-Hypopnea Index (AHI) relationship between > 5 or < 5 SBQ and RP AHI (p<0.05). BMI > 25 kg/m2 + snoring (S) + observed apnea (O) + 1 of the following: ESS > 11, hypertension (HT) or > 5 SBQ components showed sensitivity of 40% (CI95%: 37.3-43) and specificity of 95.1% (CI93.4-96.4). The performance of 5 SBQ components with regard to gender and empirical CPAP therapy was; (women vs. men): AUC-ROC 0.625 (CI95%: 0.599-0.651) vs. 0.70 (CI95%: 0.68-0.72), p<0.01, respectively. Conclusions STOP-BANG and ESS made it possible to indicate CPAP reliably (low rate of false-positive results) in 20-40% of patients who needed such therapy according to clinical history and RP results. These clinical criteria performed better in male.