Eosinophil autofluorescence and its use in isolation and analysis of human eosinophils using flow microfluorometry

Unstained human eosinophils exhibit unusually bright autofluorescence, which allows them to be distinguished from other leukocytes using fluorescence microscopy. Eosinophil fluorescence is associated with the cytoplasmic granules of the cells. Eosinophil granule extracts, containing an as-yet-undefined eosinophil fluorescence factor, exhibited excitation maxima at 370 nm and 450 nm, with maximum emission at 520 nm. Eosinophils adhering to opsonized parasites in vitro deposit fluorescent material onto the parasite surface. Eosinophil fluorescence was of sufficient intensity to allow the preparation of viable, highly enriched (greater than or equal to 98%), eosinophil suspensions from peripheral blood of normal and eosinophilic donors using a fluorescence- activated cell sorter. Quantitative studies of eosinophil autofluorescence were performed using flow microfluorometry. Fluorescence intensity of blood eosinophils from normal volunteers and eosinophilic patients varied inversely with the log of the donor's absolute eosinophil count regardless of clinical diagnosis.     

Renal cell carcinoma metastases to gallbladder

A 74-year old man underwent a radical cholecystectomy for presumed gallbladder cancer. The histology of the resected specimen in fact revealed the lesion to be metastatic renal cell carcinoma from his resected right nephrectomy performed 14 years previously.     

Hydroxyurea-induced augmentation of fetal hemoglobin production in patients with sickle cell anemia

Five patients with sickle cell anemia were treated with hydroxyurea (HU), in hopes of augmenting their production of fetal hemoglobin. Laboratory responses in two patients treated for more than 2 years were encouraging and there were suggestions of clinical improvement. Long- term HU therapy should be considered for severely affected adults with sickle cell anemia who are willing to accept what is probably a small risk of carcinogenesis. Preliminary chromosomal analysis and knowledge of the clastogenic properties of HU suggest that conception and pregnancy should be avoided. Pharmacokinetic studies will probably be necessary to adjust individual dosage schedules so that cytotoxicity is avoided. F cell responses can be seen in 2 to 3 weeks if the HU dose is optimal, but establishment of a large number of F cells in the circulation may take a month or longer.     

Pre-B acute lymphoblastic leukemia cells may induce T-cell anergy to alloantigen

Even if neoplastic cells express tumor associated antigens they still may fail to function as antigen presenting cells (APC) if they lack expression of one or more molecules critical for the induction of productive immunity. These cellular defects can be repaired by physiologic activation, transfection, or fusion of tumor cells with professional APC. Although such defects can be repaired, antitumor specific T cells may still fail to respond in vivo if they may have been tolerized. Here, human pre-B cell acute lymphoblastic leukemia (pre-B ALL) was used as a model to determine if primary human tumor cells can function as alloantigen presenting cells (alloAPC) or alternatively whether they induce anergy. In the present report, we show that pre-B cell ALL express alloantigen and adhesion molecules but uniformly lack B7-1 (CD80) and only a subset express B7-2 (CD86). Pre-B ALL cells are inefficient or ineffective alloAPC and those cases that lack expression of B7-1 and B7-2 also induce alloantigen specific T- cell unresponsiveness. Under these circumstances, T-cell unresponsiveness could be prevented by physiologic activation of tumor cells via CD40, cross-linking CD28, or signaling through the common gamma chain of the interleukin-2 receptor on T cells. Taken together, these results suggest that pre-B ALL may be incapable of inducing clinically significant T-cell-mediated antileukemia responses. This defect may be not only due to their inability to function as APC, but also due to their potential to induce tolerance. Attempts to induce clinically significant antitumor immune responses may then require not only mechanisms to repair the antigen presenting capacity of the tumor cells, but also reversal of tolerance.     

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.     

Downregulation of proteins involved in the endoplasmic reticulum stress response and Nrf2-ARE signaling in lymphoblastoid cells of spinocerebellar ataxia type 17

Spinocerebellar ataxia type 17 (SCA17) is caused by CAG repeat expansion in the TATA-box binding protein gene. Studies of several polyglutamine (polyQ) expansion diseases have suggested that the expanded polyQ proteins misfold and induce oxidative stress to contribute to cell death. Substantial deficits in peripheral tissues including lymphocytes have been shown and these peripheral abnormalities could also be found in neurons possessing polyQ disease proteins. In this study, we used a lymphoblastoid cell model to investigate the functional implication of SCA17 expanded alleles and assess the potential therapeutic strategies that may ameliorate the effects of expanded polyQ. Proteomics studies of patient/control pairs including two-dimensional (2-D) gel electrophoresis, mass spectrometry and immunoblotting were conducted. A total of 8 proteins with reduced expression changes greater than 1.3-fold were identified, including previously reported HSPA5 and HSPA8. Among 6 proteins further semi-quantified by immunoblotting and real-time PCR, the reduced expression of HYOU1, PDIA3, P4HB, NQO1 and HMOX1 was confirmed. Treatment with resveratrol and genipin up-regulated NQO1 and HMOX1 expression and reduced oxidative stress in patients’ lymphoblastoid cells. The results illustrate downregulation of proteins involved in the endoplasmic reticulum stress response (HYOU1, HSPA5, PDIA3, and P4HB) and Nrf2-ARE signaling (NQO1 and HMOX1) in SCA17 lymphoblastoid cells. Compounds increasing anti-oxidative activity such as resveratrol and genipin may serve as a potential therapeutic strategy for SCA17.     

INDIVIDUALISATION OF HIV THERAPY: THE CLINICIAN'S PERSPECTIVE

SUMMARY Assessment of clinical and laboratory markers of HIV infection may be used to individualise antiretroviral therapy. Data suggest that measures of viral load may be of considerable value as both a baseline and dynamic therapy marker, making these tools particularly useful in driving therapeutic decisions. Similarly, in-vitro data regarding intracellular pharmacokinetics and activity, resistance patterns and potential synergy of antiretroviral agents may be used to guide selection of optimal treatment regimens in clinical practice.     

Structural requirements of platelet chemokines for neutrophil activation

Using recombinantly expressed proteins and synthetic peptides, we examined the structural/functional features of the platelet chemokines, neutrophil-activating peptide-2 (NAP-2) and platelet factor 4 (PF4); that were important in their activation of neutrophils. Previous studies with the chemokine interleukin-8 (IL-8) had shown that the N- terminal region preceding the first cysteine residue was critical in defining neutrophil-activating properties. We examined whether NAP-2 and PF4 had similar structural requirements. In the Ale-glu-leu-arg (AELR) N-terminus of NAP-2, substitution of E or R abolished Ca2+ mobilization and elastase secretion. Unlike the parent molecule PF4, AELR/PF4, the hybrid formed by replacing the N-terminal sequence of PF4 before the first cysteine residue with the homologous sequence of NAP- 2, stimulated Ca2+ mobilization and elastase secretion. Furthermore, the effect of amino acid substitutions in the ELR motif differed from those seen with NAP-2 in that conserved substitutions of E or R in NAP- 2 abolished activity, but only reduced neutrophil activation in the hybrid. These studies show that just as with IL-8, the N-termini of NAP- 2 and PF4 are critical for high-level neutrophil-activating function. Desensitization studies provided information on receptor binding. NAP- 2, which binds almost exclusively to the type 2 IL-8 receptor (IL-8R), did not desensitize neutrophils to activation by IL-8 because IL-8 could bind to and activate via both type 1 and 2 IL-8R. AELR/PF4 appears to bind to both types of receptors because it desensitized neutrophils to NAP-2 activation; but was not desensitized by NAP-2, and because it desensitized to and was desensitized by IL-8. Thus, although NAP-2 and AELR/PF4 share approximately 60% amino acid homology, they have different receptor affinities. Studies were performed to define the role of the C-termini of these platelet chemokines in receptor binding. Heparin and a monoclonal antibody specific for the heparin- binding domain of PF4 both inhibited Ca2+ mobilization and elastase release, further suggesting that the C-terminus of these chemokines is important in receptor binding. Synthetic NAP-2(51-70) failed to mobilize Ca2+, whereas PF4(47-70) and PF4(58-70) induced Ca2+ mobilization and secretion of elastase at high concentrations. Pertussis toxin inhibited neutrophil activation by 40% to 50%, establishing a role for G-protein-coupled receptors such as the IL-8Rs in activation by the PF4 C-terminal peptides.(ABSTRACT TRUNCATED AT 400 WORDS)     

Association between PARK16 and Parkinson's disease in the Han Chinese population: a meta-analysis

PARK16 was reported to alter the risk for Parkinson's disease (PD) in the Japanese population. However, its role in Han Chinese PD patients has not been well established. Herein, we investigated the effect of 4 single-nucleotide polymorphisms (SNPs) within the PARK16 locus, including rs823128, rs947211, rs823156, and rs11240572, on the risk of PD by genotyping 497 Taiwanese patients with PD and 500 age-matched control subjects. The results were then meta-analyzed with available genetic association studies in the same population. The meta-analysis showed that PD patients demonstrated a lower frequency of the rs823128 G allele (11.93%) than control subjects (14.04%; odds ratio [OR] 0.83, 95% confidence interval [CI] 0.72–0.96, p = 0.010). The frequency of the rs947211 A allele (40.35%) in PD patients was lower than in control subjects (43.01%; OR 0.90, 95% CI 0.80–0.99, p = 0.047). The rs823156 G allele was less frequently seen in PD patients (17.32%) than in control subjects (21.35%; OR 0.77, 95% CI 0.69–0.86, p < 0.001). A lower frequency of the rs11240572 A allele was found in PD patients (14.01%) than in control subjects (17.66%; OR 0.76, 95% CI 0.66–0.88, p < 0.001). Our results indicate a robust protective effect of PARK16 in Han Chinese PD patients. Functional approaches are needed to elucidate the effects of these SNPs on the regulation of gene expression.