CD18-dependent and L-selectin-dependent neutrophil emigration is diminished in neonatal rabbits

Human neonatal neutrophils manifest decreases in mobility, adherence, and emigration compared with adult neutrophils that may contribute to the increased susceptibility of neonates to infection. In a developmental rabbit model, we show a reduced ability of neutrophils from 1-day-old rabbit pups to emigrate to inflamed peritoneium (3.7 +/- 0.35 x 10(6) neutrophils/mL peritoneal exudate) compared with 14-day- old (8.5 +/- 0.7 x 10(6)/mL) and adult rabbits (9.4 +/- 1.4 x 10(6) mL, P < .05) despite significantly increased blood neutrophil counts. Because the reductions in functional Mac-1 (CD11b/CD18) as well as the amount of surface L-selectin are hypothesized to be primarily responsible for the differences in human neonatal neutrophil mobility, we examined CD11b/CD18 and L-selectin in our model. Using flow cytometric analysis we found that similar to human neonates, neutrophils from 1-day-old rabbit pups had 57% of adult rabbit levels of L-selectin and, in contrast with adults, failed to show significant decreases in L-selectin after chemotactic stimulation. In addition, neutrophils from 1-day-old pups compared with adults showed a significantly diminished capacity to upregulate CD11b/CD18 after chemotactic stimulation in vitro, or after emigration to the inflamed peritoneum. Systemic administration of anti-L-selectin monoclonal antibody (MoAb) resulted in significant reduction in peritoneal neutrophils in adult (47%, P < .05) and 14-day-old rabbits (47%, P < .05), but was without effect in 1-day-old rabbits. Administration of anti-CD18 MoAb resulted in significant reduction in peritoneal neutrophil accumulation in all age groups though less in 1 day and 14 day (58% and 65%, respectively) than in adults (91%, P < .05). Only in the 14-day-old rabbits was there an additive effect of anti-L-selectin and anti-CD18 MoAbs compared with anti-CD18 alone (84% v 65%, P < .05). The findings in this in vivo rabbit model support the hypothesis that the previously described in vitro defects in human neonatal L-selectin and CD11b/CD18 may be major contributors to human neonatal inflammatory deficits.     

Quality control of multidrug resistance assays in adult acute leukemia: correlation between assays for P-glycoprotein expression and activity

We have compared multiple assays for the P-glycoprotein (Pgp/MDR1) phenotype in fresh and thawed adult acute leukemia to validate and quantitate measures for the expression and function of Pgp. The results are related to the Pgp-expressing KB8 and KB8-5 call lines. The most sensitive assay was the measurement of modulation of the rhodamine 123 (R123) fluorescence by 2 micromol/L PSC833, followed by the modulation of the probe calcein-AM. We also found a good intralaboratory and interlaboratory correlation between the values of the R123/PSC833 assay for fresh as well as thawed samples. In addition, the affects of PSC833 on 3H-daunorubicin (DNR) accumulation, DNR fluorescence, and 3H- vincristine accumulation were very similar. The correlation between the DNR/PSC833 and R123/PSC833 test was r = .86 (N = 51). The modulation of drug accumulation by 8 micromol/L verapamil was the some as the PSC833 effect for DNR (117%, N = 21), but was higher for vincristine in every single case (161% v 121%, N = 22; P< .001), indicating additional verapamil effects, not related to Pgp. The correlation of the staining of viable cells for Pgp with the monoclonal antibody MRK16 was r = .77 (N = 52) for the R123/PSC833 functional test and r = .84 (N = 50) for the DNR/PSC833 test. From these results it could be calculated that a maximal increase of the mean DNR accumulation of about 50% can be achieved by blocking Pgp pump activity with PSC833 in leukemic blast samples with the highest mean Pgp expression. Subpopulations of blast calls with higher Pgp activity are likely to be present. Their relevance has to be studied further. The methods outlined here allow the reliable, quantitative monitoring of the Pgp/MDR1 phenotype in leukemias in multicentered, clinical Pgp modulation studies.     

Establishment and characterization of an Epstein-Barr virus transformed cell line with strong phagocytic activity

An immunoglobulin M (IgM)-positive cell line, Ms 28, apparently spontaneously transformed by Epstein-Barr virus (EBV) was established from peripheral blood cells of a patient with immature myeloblastic leukemia. It has been characterized according to phenotype, cytochemistry, and membrane antigen pattern. The cell line expresses lymphoid markers like CD 19, CD 22, and CD 30 and synthesizes and secretes IgM. Monocyte markers CD 11c, CD 14, and CD 15 are absent. Neither interleukin-1 (IL-1), nor tumor necrosis factor (TNF-alpha) are produced. But Ms 28 cells show strong phagocytic activity and engulf Latex particles and sheep RBCs (SRBCs) that need not to be opsonized. The phagocytic activity can be inhibited by chloroquine. Both phagocytosis and EBV nuclear-antigen (EBNA) expression can be observed in one and the same cell. Ms 28 cells might be useful to study immunologic activities like antigen processing and presentation.     

PET/CT and MR imaging biomarker of lipid-rich plaques using [Cu]-labeled scavenger receptor (CD68-Fc)

Continued uptake of modified low-density lipoproteins (LDL) by the scavenger receptor, CD68, of activated macrophages is a crucial process in the development of atherosclerotic plaques and leads to the formation of foam cells. Eight-weeks-old male Apolipoprotein E-deficient (ApoE^-/-) mice (n=6) were fed a high-fat diet for 12 weeks. C57BL/6J wildtype (WT) mice served as controls (n=6). Positron emission tomography (PET) with an acquisition time of 1800s (NanoPET/CT scanner; Mediso, Hungary & Bioscan, USA) was carried out 24h after intravenous tail vein administration of 50µl ⁶⁴Cu-CD68-Fc (~20-30µg labeled protein/mouse containing approximately 10-12MBq ⁶⁴Cu-CD68-Fc per mouse). Three days after PET/CT, all mice received an intravenous administration of 0.2 mmol/kg body weight of a gadolinium-based elastin-binding contrast agent to assess plaque burden and vessel wall remodeling. Two hours after injection, mice were imaged in a 3T clinical MR scanner (Philips Healthcare, Best, NL) using a dedicated single loop surface coil (23mm). Enhanced ⁶⁴Cu-CD68-Fc uptake was found in the aortic arches of ApoE^-/- compared to WT mice (ApoE^-/- mice:10.5±1.5Bq/cm³ vs. WT mice: 2.1±0.3Bq/cm³; P=0.002). Higher gadolinium-based elastin-binding contrast agent uptake was also detected in the aortic arch of ApoE^-/- compared to WT mice using R¹ maps (R¹=1.47±0.06 s^-1 vs. 0.92±0.05 s^-1; P <0.001). Radiolabeled scavenger receptor (⁶⁴Cu-CD68-Fc) may help to target foam cell rich plaques with high content of oxidized LDL. This novel imaging biomarker tool may have potential to identify unstable plaques and for risk stratification.     

High viral suppression and low attrition in healthy HIV-infected patients initiated on ART with CD4 above 500 cells/µL in a program setting in Uganda

BackgroundThe World Health Organization recommends antiretroviral therapy (ART) for all HIV-infected patients at all CD4 counts. However, there are concerns that asymptomatic patients may have poorer viral suppression and high attrition.ObjectivesWe sought to determine attrition and viral suppression among healthy HIV-infected patients initiated on ART in program settings.MethodsThis cross-sectional study enrolled ART-experienced patients attending two PEPFAR-supported, high-volume clinics in Kampala, Uganda. Eligible patients were >18 years and had completed at least six months on ART. Participants were interviewed on socio-demographics, ART history and plasma viral load (VL) determined using Abbott Real-time. Predictors of viral suppression (<75 copies/ml) were determined using multivariate logistic regression.ResultsOverall, 267 participants were screened, 228 were eligible and 203 (89%) retained in care (visit within 90 days). Of the 203 participants, 115 (56.7%) were key-populations. Viral suppression was achieved in 173 patients (85%; 95% CI, 80.3%–90.1%). The factors associated with viral suppression were prior VL tests (AOR 6.98; p-value <0.001) and receiving care from a general clinic (AOR 5.41; p=0.009).ConclusionAsymptomatic patients initiated on ART with high baseline CD4 counts, achieve high viral suppression with low risk of attrition. VL monitoring and clinic type are associated with viral suppression.     

Multiple primary malignancies in patients with Merkel cell carcinoma1

Background Merkel cell carcinoma (MCC) is a rare malignant cutaneous tumour, the incidence of which is increasing. Second malignancies have been reported to occur with high incidence in these patients. Objectives We report the rate and nature of multiple malignancies in patients with MCC treated over a 10 year period in Addenbrooke’s Hospital in Cambridge, United Kingdom, as well as the temporal relationship of these additional malignancies to the diagnosis of MCC. Results The 27 patients had an approximately equal sex incidence with a median age at diagnosis of 79 years. Seventy percent (n=19) of patients had a second primary malignant tumour; and 7 of these patients had two or more tumours in addition to the MCC. Eighteen patients had additional cutaneous malignancies: melanoma, squamous cell carcinoma and basal cell carcinoma, and 8 patients presented non‐cutaneous malignancy including colorectal, haematological and breast tumours. Of the 28 additional tumours in our patients, half were diagnosed prior to presentation of MCC, 32% within 6 months of diagnosis, and 18% between 6 months and 3 years after diagnosis. Possible reasons for the high rate of additional tumours in this population are discussed. Conclusions Our figures reflect a higher incidence of multiple malignancies in those with Merkel cell tumour than has previously been reported. This has important implications for the care and surveillance of these patients.