Synthesis of two potential NK<Subscript>1</Subscript>-receptor ligands using [1-<Superscript>11</Superscript>C]ethyl iodide and [1-<Superscript>11</Superscript>C]propyl iodide and initial PET-imaging

Background

The previously validated NK₁-receptor ligand [O-methyl-¹¹C]GR205171 binds with a high affinity to the NK₁-receptor and displays a slow dissociation from the receptor. Hence, it cannot be used in vivo for detecting concentration changes in substance P, the endogenous ligand for the NK₁-receptor. A radioligand used for monitoring these changes has to enable displacement by the endogenous ligand and thus bind reversibly to the receptor. Small changes in the structure of a receptor ligand can lead to changes in binding characteristics and also in the ability to penetrate the blood-brain barrier. The aim of this study was to use carbon-11 labelled ethyl and propyl iodide with high specific radioactivity in the synthesis of two new and potentially reversible NK₁-receptor ligands with chemical structures based on [O-methyl-¹¹C]GR205171.

Methods

[1-¹¹C]Ethyl and [1-¹¹C]propyl iodide with specific radioactivities of 90 GBq/μmol and 270 GBq/μmol, respectively, were used in the synthesis of [O-methyl-¹¹C]GR205171 analogues by alkylation of O-desmethyl GR205171. The brain uptake of the obtained (2S,3S)-N-(1-(2- [1-¹¹C]ethoxy-5-(3-(trifluoromethyl)-4H-1,2,4-triazol-4-yl)phenyl)ethyl)-2-phenylpiperidin-3-amine (I) and (2S,3S)-2-phenyl-N-(1-(2- [1-¹¹C]propoxy-5-(3-(trifluoromethyl)-4H-1,2,4-triazol-4-yl)phenyl)ethyl)piperidin-3-amine (II) was studied with PET in guinea pigs and rhesus monkeys and compared to the uptake of [O-methyl-¹¹C]GR205171.

Results

All ligands had similar uptake distribution in the guinea pig brain. The PET-studies in rhesus monkeys showed that (II) had no specific binding in striatum. Ligand (I) had moderate specific binding compared to the [O-methyl-¹¹C]GR205171. The ethyl analogue (I) displayed reversible binding characteristics contrary to the slow dissociation rate shown by [O-methyl-¹¹C]GR205171.

Conclusion

The propyl-analogue (II) cannot be used for detecting changes in NK₁-ligand levels, while further studies should be performed with the ethyl-analogue (I).

     

Monitoring of CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T-Cell Responses After Dendritic Cell-Based Immunotherapy Using CFSE Dye Dilution Analysis

CFSE dye dilution analysis and [³H] thymidine incorporation were used side by side to assess proliferative responses of peripheral blood mononuclear cells (PBMCs) after vaccination of renal cell carcinoma patients (n=6) with antigen-loaded dendritic cells. Immune responses against the control antigen keyhole limpet hemocyanin (KLH) were induced in all patients. While [³H] thymidine incorporation revealed a 4 to 977-fold increase in KLH-induced proliferation (mean: 209-fold), CFSE-labeling experiments demonstrated that the KLH-responsive population of postvaccination PBMCs represented 7–53% (mean: 23%). Combining CFSE-labeling with T-cell subset analysis confirmed the presence of CD4⁺ KLH-reactive T cells but also revealed a substantial population of CD8⁺ KLH-reactive T cells in one patient as well as minor populations of CD8⁺ KLH-reactive T cells in three other patients. Our data indicate that CFSE dye dilution analysis is a valuable tool for immune monitoring after dendritic cell vaccination.     

Loss of serotonin 2A receptors exceeds loss of serotonergic projections in early Alzheimer's disease: a combined [C]DASB and [F]altanserin-PET study

In patients with Alzheimer's disease (AD), postmortem and imaging studies have revealed early and prominent reductions in cerebral serotonin 2A (5-HT_2A) receptors. To establish if this was due to a selective disease process of the serotonin system, we investigated the cerebral 5-HT_2A receptor and the serotonin transporter binding, the latter as a measure of serotonergic projections and neurons. Twelve patients with AD (average Mini Mental State Examination [MMSE]: 24) and 11 healthy age-matched subjects underwent positron emission tomography (PET) scanning with [¹⁸F]altanserin and [¹¹C]N,N-Dimethyl-2-(2-amino-4-cyanopheylthio)benzylamine ([¹¹C]DASB). Overall [¹⁸F]altanserin binding was markedly reduced in AD by 28%-39% (p = 0.02), whereas the reductions in [¹¹C]DASB binding were less prominent and mostly insignificant, except for a marked reduction of 33% in mesial temporal cortex (p = .0005). No change in [¹¹C]DASB binding was found in the midbrain. We conclude that the prominent reduction in neocortical 5-HT_2A receptor binding in early AD is not caused by a primary loss of serotonergic neurons or their projections.     

Metabotropic glutamate receptor type 5 in levodopa-induced motor complications

Metabotropic glutamate receptors type 5 (mGluR5) are implicated in regulation of synaptic plasticity and learning, and were the focus of our investigation in human Parkinson's disease (PD) patients with dyskinesias and wearing-off, and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys with dyskinesias. Using the selective mGluR5 ligand [³H]ABP688 autoradiography, we measured mGluR5 in brain slices from 11 normal and 14 PD patients and from MPTP monkeys, in relation to motor complications (dyskinesias and wearing-off) associated with treatment with l-dopa. In 16 monkeys with a bilateral MPTP lesion and four controls, [³H]ABP688 specific binding was elevated in the striatum of dyskinetic l-dopa-treated MPTP monkeys but not in MPTP monkeys without dyskinesias compared to controls. PD patients with motor complications (either dyskinesias or wearing-off) had higher [³H]ABP688 specific binding compared to those without motor complications and controls in putamen, external and internal globus pallidus. Elevated glutamatergic transmission as measured with increased mGluR5 specific binding was associated with motor complications and its antagonism could be targeted for their treatment.     

Complement Split Products C3a and C4a in Chronic Lyme Disease

Complement split products C3a and C4a are reportedly elevated in patients with acute Lyme disease. We have now examined these immunologic markers in patients with chronic Lyme disease compared to appropriate disease controls. The study population consisted of 29 healthy controls, 445 patients with chronic Lyme disease, 11 patients with systemic lupus erythematosus (SLE) and six patients with AIDS. The Lyme disease patients were divided according to predominant musculoskeletal symptoms (324 patients) or predominant neurologic symptoms (121 patients). C3a and C4a levels were measured by radioimmunoassay. All patients with chronic Lyme disease and AIDS had normal C3a levels compared to controls, whereas patients with SLE had significantly increased levels of this marker. Patients with predominant musculoskeletal symptoms of Lyme disease and AIDS patients had significantly increased levels of C4a compared to either controls, patients with predominant neurologic symptoms of Lyme disease or SLE patients. Response to antibiotic therapy in chronic Lyme disease was associated with a significant decrease in the C4a level, whereas lack of response was associated with a significant increase in this marker. In contrast, AIDS patients had persistently increased C4a levels despite antiretroviral therapy. Lyme patients with positive single-photon emission computed tomographic (SPECT) scans had significantly lower C4a levels compared to Lyme patients with normal SPECT scan results. Patients with predominant musculoskeletal symptoms of Lyme disease have normal C3a and increased C4a levels. This pattern differs from the increase in both markers seen in acute Lyme disease, and C4a changes correlate with the response to therapy in chronic Lyme disease. C4a appears to be a valuable immunologic marker in patients with persistent symptoms of Lyme disease.     

Addition of vasopressin synthetic analogue [V<Superscript>4</Superscript>Q<Superscript>5</Superscript>]dDAVP to standard chemotherapy enhances tumour growth inhibition and impairs metastatic spread in aggressive breast tumour models

[V⁴Q⁵]dDAVP is a novel 2nd generation vasopressin analogue with robust antitumour activity against metastatic breast cancer. We recently reported that, by acting on vasopressin V2r membrane receptor present in tumour cells and microvascular endothelium, [V⁴Q⁵]dDAVP inhibits angiogenesis and metastatic progression of the disease without overt toxicity. Despite chemotherapy remaining as a primary therapeutic option for aggressive breast cancer, its use is limited by low selectivity and associated adverse effects. In this regard, we evaluated potential combinational benefits by adding [V⁴Q⁵]dDAVP to standard-of-care chemotherapy. In vitro, combination of [V⁴Q⁵]dDAVP with sub-IC₅₀ concentrations of paclitaxel or carmustine resulted in a cooperative inhibition of breast cancer cell growth in comparison to single-agent therapy. In vivo antitumour efficacy of [V⁴Q⁵]dDAVP addition to chemotherapy was first evaluated using the triple-negative MDA-MB-231 breast cancer xenograft model. Tumour-bearing mice were treated with i.v. injections of [V⁴Q⁵]dDAVP (0.3 μg/kg, thrice weekly) in combination with weekly cycles of paclitaxel (10 mg/kg i.p.). After 6 weeks of treatment, combination regimen resulted in greater tumour growth inhibition compared to monotherapy. [V⁴Q⁵]dDAVP addition was also associated with reduction of local aggressiveness, and impairment of tumour invasion and infiltration of the skin. Benefits of combined therapy were confirmed in the hormone-independent and metastatic F3II breast cancer model by combining [V⁴Q⁵]dDAVP with carmustine (25 mg/kg i.p.). Interestingly, [V⁴Q⁵]dDAVP plus cytotoxic agents severely impaired colony forming ability of tumour cells and inhibited breast cancer metastasis to lung. The present study shows that [V⁴Q⁵]dDAVP may complement conventional chemotherapy by modulating metastatic progression and early stages of microtumour establishment, and thus supports further preclinical testing of the compound for the management of aggressive breast cancer.     

The diagnostic performance of <Superscript>18</Superscript>F-FAMT PET and <Superscript>18</Superscript>F-FDG PET for malignancy detection: a meta-analysis

Background

This meta-analysis aims to compare the diagnostic performance of l-3-¹⁸F-α-methyl tyrosine (¹⁸F-FAMT) positron emission tomography (PET) and 2-deoxy-2-[¹⁸F]fluoro-d-glucose (¹⁸F-FDG) PET for malignancy detection.

Methods

The workflow of this study follows Cochrane Collaboration Guidelines of a systematic review of diagnostic test accuracy studies. An electronic search was performed for clinical diagnostic studies directly comparing ¹⁸F-FAMT and ¹⁸F-FDG PET for malignant tumors. Study quality, the risks of bias and sources of variation among studies were assessed using the QUADAS (Quality Assessment of Diagnostic Accuracy Studies) assessment tool. A separate meta-analysis was performed for diagnostic performance based on visual assessment and diagnostic cut-off values. Whenever possible, a bivariate random-effect model was used for analysis and pooling of diagnostic measures across studies.

Results

Electronic search revealed 56 peer-reviewed basic science investigations and clinical studies. Six eligible studies (272 patients) of various type of cancer were meta-analyzed. The ¹⁸F-FAMT diagnostic accuracy for malignancy was higher than ¹⁸F-FDG based on both visual assessment (diagnostic odd ratio (DOR): 8.90, 95% confidence interval (CI) [2.4, 32.5]) vs 4.63, 95% CI [1.8, 12.2], area under curve (AUC): 77.4% vs 72.8%) and diagnostic cut-off (DOR: 13.83, 95% CI [6.3, 30.6] vs 7.85, 95% CI [3.7, 16.8], AUC: 85.6% vs 80.2%), respectively. While the average sensitivity and specificity of ¹⁸F-FAMT and ¹⁸F-FDG based on visual assessment were similar, ¹⁸F-FAMT was significantly more specific than ¹⁸F-FDG (p?<?0.05) based on diagnostic cut-off values.

Conclusions

¹⁸F-FAMT is more specific for malignancy than ¹⁸F-FDG, while their sensitivity is comparable. ¹⁸F-FAMT PET is equal to ¹⁸F-FDG PET in diagnostic performance for malignancy detection in several cancer types.

     

In vivo evaluation of amyloid deposition and brain glucose metabolism of 5XFAD mice using positron emission tomography

Positron emission tomography (PET) has been used extensively to evaluate the neuropathology of Alzheimer's disease (AD) in vivo. Radiotracers directed toward the amyloid deposition such as [¹⁸F]-FDDNP (2-(1-{6-[(2-[F]Fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile) and [¹¹C]-PIB (Pittsburg compound B) have shown exceptional value in animal models and AD patients. Previously, the glucose analogue [¹⁸F]-FDG (2-[(18)F]fluorodeoxyglucose) allowed researchers and clinicians to evaluate the brain glucose consumption and proved its utility for the early diagnosis and the monitoring of the progression of AD. Animal models of AD are based on the transgenic expression of different human mutant genes linked to familial AD. The novel transgenic 5XFAD mouse containing 5 mutated genes in its genome has been proposed as an AD model with rapid and massive cerebral amyloid deposition. PET studies performed with animal-dedicated scanners indicate that PET with amyloid-targeted radiotracers can detect the pathological amyloid deposition in transgenic mice and rats. However, in other studies no differences were found between transgenic mice and their wild type littermates. We sought to investigate in 5XFAD mice if the radiotracers [¹¹C]-PIB, and [¹⁸F]-Florbetapir could quantify the amyloid deposition in vivo and if [¹⁸F]-FDG could do so with regard to glucose consumption. We found that 5XFAD animals presented higher cerebral binding of [¹⁸F]-Florbetapir, [¹¹C]-PIB, and [¹⁸F]-FDG. These results support the use of amyloid PET radiotracers for the evaluation of AD animal models. Probably, the increased uptake observed with [¹⁸F]-FDG is a consequence of glial activation that occurs in 5XFAD mice.     

Hyperpolarized 13C magnetic resonance spectroscopy detects toxin‐induced neuroinflammation in mice

Lipopolysaccharide (LPS) is a commonly used agent for induction of neuroinflammation in preclinical studies. Upon injection, LPS causes activation of microglia and astrocytes, whose metabolism alters to favor glycolysis. Assessing in vivo neuroinflammation and its modulation following therapy remains challenging, and new noninvasive methods allowing for longitudinal monitoring would be highly valuable. Hyperpolarized (HP) ¹³C magnetic resonance spectroscopy (MRS) is a promising technique for assessing in vivo metabolism. In addition to applications in oncology, the most commonly used probe of [1–¹³C] pyruvate has shown potential in assessing neuroinflammation‐linked metabolism in mouse models of multiple sclerosis and traumatic brain injury. Here, we aimed to investigate LPS‐induced neuroinflammatory changes using HP [1–¹³C] pyruvate and HP ¹³C urea. 2D chemical shift imaging following simultaneous intravenous injection of HP [1–¹³C] pyruvate and HP ¹³C urea was performed at baseline (day 0) and at days 3 and 7 post‐intracranial injection of LPS (n = 6) or saline (n = 5). Immunofluorescence (IF) analyses were performed for Iba1 (resting and activated microglia/macrophages), GFAP (resting and reactive astrocytes) and CD68 (activated microglia/macrophages). A significant increase in HP [1–¹³C] lactate production was observed at days 3 and 7 following injection, in the injected (ipsilateral) side of the LPS‐treated mouse brain, but not in either the contralateral side or saline‐injected animals. HP ¹³C lactate/pyruvate ratio, without and with normalization to urea, was also significantly increased in the ipsilateral LPS‐injected brain at 7 days compared with baseline. IF analyses showed a significant increase in CD68 and GFAP staining at 3 days, followed by increased numbers of Iba1 and GFAP positive cells at 7 days post‐LPS injection. In conclusion, we can detect LPS‐induced changes in the mouse brain using HP ¹³C MRS, in alignment with increased numbers of microglia/macrophages and astrocytes. This study demonstrates that HP ¹³C spectroscopy has substantial potential for providing noninvasive information on neuroinflammation.     

Distribution of <Superscript>3</Superscript>H-Dopamine and <Superscript>3</Superscript>H-DAGO Binding Sites in the Central Part of Rat Sinoatrial Node

Distribution of ³H-dopamine and ³H-DAGO binding sites was studied by autoradiography on semithin sections of total preparations of rat sinoatrial node. The relative density of ³H-dopamine and ³H-DAGO binding sites in the functional nucleus of the sinoatrial node was minimum and increased in the cranial and caudal directions. The level of ³H-dopamine binding in the perinodal atrial myocardium was appreciably lower (22±6%), while binding of ³H-DAGO was similar (76±16%) to that in the periarterial zone of the sinoatrial node. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 140, No. 8, pp. 210–214, August, 2005     

Para-chloro-2-[18F]fluoroethyl-etomidate: A promising new PET radiotracer for adrenocortical imaging

Introduction: [¹¹C]Metomidate ([¹¹C]MTO), the methyl ester analogue of etomidate, was developed as a positron emission tomography (PET) radiotracer for adrenocortical tumours and has also been suggested for imaging in primary aldosteronism (PA). A disadvantage of [¹¹C]MTO is the rather high non-specific binding in the liver, which impacts both visualization and quantification of the uptake in the right adrenal gland. Furthermore, the short 20-minute half-life of carbon-11 is a logistic challenge in the clinical setting.Objectives: The aim of this study was to further evaluate the previously published fluorine-18 (T_1/2=109.5 min) etomidate analogue, para-chloro-2-[¹⁸F]fluoroethyl etomidate; [¹⁸F]CETO, as an adrenal PET tracer.Methods: In vitro experiments included autoradiography on human and cynomolgus monkey (non-human primate, NHP) tissues and binding studies on adrenal tissue from NHPs. In vivo studies with [¹⁸F]CETO in mice, rats and NHP, using PET and CT/MRI, assessed biodistribution and binding specificity in comparison to [¹¹C]MTO.Results: The binding of [¹⁸F]CETO in the normal adrenal cortex, as well as in human adrenocortical adenomas and adrenocortical carcinomas, was shown to be specific, both in vitro (in humans) and in vivo (in rats and NHP) with an in vitro K_d of 0.66 nM. Non-specific uptake of [¹⁸F]CETO in NHP liver was found to be low compared to that of [¹¹C]MTO.Conclusions: High specificity of [¹⁸F]CETO to the adrenal cortex was demonstrated, with in vivo binding properties qualitatively surpassing those of [¹¹C]MTO. Non-specific binding to the liver was significantly lower than that of [¹¹C]MTO. [¹⁸F]CETO is a promising new PET tracer for imaging of adrenocortical disease and should be evaluated further in humans.     

Effects of membrane depolarization and changes in extracellular [K<Superscript>+</Superscript>] on the Ca<Superscript>2+</Superscript> transients of fast skeletal muscle fibers. Implications for muscle fatigue

Repetitive activation of skeletal muscle fibers leads to a reduced transmembrane K⁺ gradient. The resulting membrane depolarization has been proposed to play a major role in the onset of muscle fatigue. Nevertheless, raising the extracellular K⁺ ( \textK_\texto ⁺ {\text{K}}_{\text{o}}^{ + } ) concentration ( [ \textK ⁺ ]_\texto [ {\text{K}}^{ + } ]_{\text{o}} ) to 10 mM potentiates twitch force of rested amphibian and mammalian fibers. We used a double Vaseline gap method to simultaneously record action potentials (AP) and Ca²⁺ transients from rested frog fibers activated by single and tetanic stimulation (10 pulses, 100 Hz) at various [ \textK ⁺ ]_\texto [ {\text{K}}^{ + } ]_{\text{o}} and membrane potentials. Depolarization resulting from current injection or raised [ \textK ⁺ ]_\texto [ {\text{K}}^{ + } ]_{\text{o}} produced an increase in the resting [Ca²⁺]. Ca²⁺ transients elicited by single stimulation were potentiated by depolarization from −80 to −60 mV but markedly depressed by further depolarization. Potentiation was inversely correlated with a reduction in the amplitude, overshoot and duration of APs. Similar effects were found for the Ca²⁺ transients elicited by the first pulse of 100 Hz trains. Depression or block of Ca²⁺ transient in response to the 2nd to 10th pulses of 100 Hz trains was observed at smaller depolarizations as compared to that seen when using single stimulation. Changes in Ca²⁺ transients along the trains were associated with impaired or abortive APs. Raising [ \textK ⁺ ]_\texto [ {\text{K}}^{ + } ]_{\text{o}} to 10 mM potentiated Ca²⁺ transients elicited by single and tetanic stimulation, while raising [ \textK ⁺ ]_\texto [ {\text{K}}^{ + } ]_{\text{o}} to 15 mM markedly depressed both responses. The effects of 10 mM \textK_\texto ⁺ {\text{K}}_{\text{o}}^{ + } on Ca²⁺ transients, but not those of 15 mM \textK_\texto ⁺ {\text{K}}_{\text{o}}^{ + } , could be fully reversed by hyperpolarization. The results suggests that the force potentiating effects of 10 mM \textK_\texto ⁺ {\text{K}}_{\text{o}}^{ + } might be mediated by depolarization dependent changes in resting [Ca²⁺] and Ca²⁺ release, and that additional mechanisms might be involved in the effects of 15 mM \textK_\texto ⁺ {\text{K}}_{\text{o}}^{ + } on force generation.     

Donepezil suppresses intracellular Ca<Superscript>2+</Superscript> mobilization through the PI3K pathway in rodent microglia

Background

Microglia are resident innate immune cells which release many factors including proinflammatory cytokines or nitric oxide (NO) when they are activated in response to immunological stimuli. Pathophysiology of Alzheimer’s disease (AD) is related to the inflammatory responses mediated by microglia. Intracellular Ca²⁺ signaling is important for microglial functions such as release of NO and cytokines. In addition, alteration of intracellular Ca²⁺ signaling underlies the pathophysiology of AD, while it remains unclear how donepezil, an acetylcholinesterase inhibitor, affects intracellular Ca²⁺ mobilization in microglial cells.

Methods

We examined whether pretreatment with donepezil affects the intracellular Ca²⁺ mobilization using fura-2 imaging and tested the effects of donepezil on phagocytic activity by phagocytosis assay in rodent microglial cells.

Results

In this study, we observed that pretreatment with donepezil suppressed the TNFα-induced sustained intracellular Ca²⁺ elevation in both rat HAPI and mouse primary microglial cells. On the other hand, pretreatment with donepezil did not suppress the mRNA expression of both TNFR1 and TNFR2 in rodent microglia we used. Pretreatment with acetylcholine but not donepezil suppressed the TNFα-induced intracellular Ca²⁺ elevation through the nicotinic α7 receptors. In addition, sigma 1 receptors were not involved in the donepezil-induced suppression of the TNFα-mediated intracellular Ca²⁺ elevation. Pretreatment with donepezil suppressed the TNFα-induced intracellular Ca²⁺ elevation through the PI3K pathway in rodent microglial cells. Using DAF-2 imaging, we also found that pretreatment with donepezil suppressed the production of NO induced by TNFα treatment and the PI3K pathway could be important for the donepezil-induced suppression of NO production in rodent microglial cells. Finally, phagocytosis assay showed that pretreatment with donepezil promoted phagocytic activity of rodent microglial cells through the PI3K but not MAPK/ERK pathway.

Conclusions

These suggest that donepezil could directly modulate the microglial function through the PI3K pathway in the rodent brain, which might be important to understand the effect of donepezil in the brain.

     

Immunological role of CD4<Superscript>+</Superscript>CD28<Superscript>null</Superscript> T lymphocytes,natural killer cells,and interferon-gamma in pediatric patients with sickle cell disease: relation to disease severity and response to therapy

Sickle cell disease (SCD) is associated with alterations in immune phenotypes. CD4⁺CD28^null T lymphocytes have pro-inflammatory functions and are linked to vascular diseases. To assess the percentage of CD4⁺CD28^null T lymphocytes, natural killer cells (NK), and IFN-gamma levels, we compared 40 children and adolescents with SCD with 40 healthy controls and evaluated their relation to disease severity and response to therapy. Patients with SCD steady state were studied, focusing on history of frequent vaso-occlusive crisis, hydroxyurea therapy, and IFN-gamma levels. Analysis of CD4⁺CD28^null T lymphocytes and NK cells was done by flow cytometry. Liver and cardiac iron overload were assessed. CD4⁺CD28^null T lymphocytes, NK cells, and IFN-gamma levels were significantly higher in patients than controls. Patients with history of frequent vaso-occlusive crisis and those with vascular complications had higher percentage of CD4⁺CD28^null T lymphocytes and IFN-gamma while levels were significantly lower among hydroxyurea-treated patients. CD4⁺CD28^null T lymphocytes were positively correlated to transfusional iron input while these cells and IFN-gamma were negatively correlated to cardiac T2* and duration of hydroxyurea therapy. NK cells were correlated to HbS and indirect bilirubin. Increased expression of CD4⁺CD28^null T lymphocytes highlights their role in immune dysfunction and pathophysiology of SCD complications.     

Elevated circulating CD14<Superscript>low</Superscript>CD16<Superscript>+</Superscript> monocyte subset in primary biliary cirrhosis correlates with liver injury and promotes Th1 polarization

Primary biliary cirrhosis (PBC) is a progressive autoimmune liver disease in which monocytes/macrophages infiltration and skewed T helper type (Th) 1 and Th17 cell responses participate in the development of the disease. Human peripheral blood monocytes are heterogeneous and can be divided into classical CD14^highCD16^?, intermediate CD14^highCD16⁺, and nonclassical CD14^lowCD16⁺ monocyte subsets. Compared to classical monocytes, CD16⁺ monocytes are generally termed pro-inflammatory monocytes and play an important pathogenic role in autoimmune diseases. However, little is known about the immunophenotype and immunopathogenic role of peripheral blood CD16⁺ monocytes in PBC. Thus, we investigated the phenotype and function of these circulating monocyte subsets from PBC patients. The frequencies of circulating CD14^highCD16⁺ and CD14^lowCD16⁺ subpopulation were increased in disease compared with healthy controls. Among them, CD14^lowCD16⁺ monocyte subset positively correlated with disease progress, liver damage indicators and serum C-reactive protein, respectively. Furthermore, the frequencies of Th1 and Th17 cells were upregulated and CD14^lowCD16⁺ monocyte subset was also positively associated with Th1 cell frequency in PBC. Using a vitro coculture model, we further found that CD14^lowCD16⁺ monocytes promoted Th1 cell polarization compared to classical monocytes. Interleukin-12 (IL-12) and direct contact of patient CD4⁺T cell and CD14^lowCD16⁺ monocytes, were responsible for CD14^lowCD16⁺ monocytes promotion of Th1 cells polarization in PBC. Our study demonstrated that the enhanced CD14^lowCD16⁺ monocyte subset participated in fostering liver damage and inflammatory responses, and promoted Th1 cells skewing in PBC.     

Expression of p21<Superscript>cip1</Superscript>, p27<Superscript>kip1</Superscript>, and p16<Superscript>INk4a</Superscript> Cyclin-Dependent Kinase Inhibitors in Papillary Thyroid Carcinoma: Correlation with Clinicopathological Factors

In a variety of human malignancies, aberrant expression of proteins involved in the control of cell-cycle progression has been reported. In this study, p21^cip1, p27^kip1, and p16^INk4a cyclin-dependent kinase inhibitors were analyzed to evaluate their usefulness in clinical management of papillary thyroid carcinoma (PTC). Archived material derived from 46 cases of PTC was analyzed immunohistochemically. Protein expression was ascertained on tissue microarrays, and results were correlated with clinicopathological features of the patients. Positive immunostaining was observed in 14 (30,4%) p21^cip1, 26 (56,5%) p27^kip1, and 14 (30,4%) p16^INk4a cases. No significant correlation between p21^cip1 or p27^kip1 and clinical factors was found. In contrast, p16^INk4a expression showed a significant correlation with initial extension of the disease. Therefore, 45.8% of patients with loco-regional extension were p16^INk4a positive, whereas overexpression was only seen in 15.7% of cases with intrathyroid disease (p < 0.05). Moreover, all patients with simultaneous p16^INk4a positivity and lack of p27^kip1 staining (four patients) presented lymph node metastases. In contrast, only 12 (28.5%) of the remaining patients showed lymph node tumor involvement. In conclusion, p16^INk4a expression suggests extrathyroid neck extension of PTC. This effect is enhanced when p27^kip1 is negative. We think that their analysis by immunohistochemistry could be useful in the management of patients with PTC.     

Pre-treatment and post-treatment assessment of the C6 test in patients with persistent symptoms and a history of Lyme borreliosis

It was recently reported that antibody to C₆, a peptide that reproduces an invariable region of the VlsE lipoprotein of Borrelia burgdorferi, declined in titer by a factor of four or more in a significant proportion of patients after successful antibiotic treatment of acute localized or disseminated Lyme borreliosis. The present study evaluated the C₆ test as a predictor of therapy outcome in a population of patients with post-treatment Lyme disease syndrome. The serum specimens tested were from patients with well-documented, previously treated Lyme borreliosis who had persistent musculoskeletal or neurocognitive symptoms. All of the patients had participated in a recent double-blind, placebo-controlled antibiotic trial in which serum samples were collected at baseline and 6 months thereafter, i.e. 3 months following treatment termination. In this patient population no correlation was found between a decline of C₆ antibody titer of any magnitude and treatment or clinical outcome. Antibodies to C₆ persisted in these patients with post-treatment Lyme disease syndrome following treatment, albeit at a markedly lower prevalence and titer than in untreated patients with acute disseminated Lyme disease. The results indicate that C₆ antibody cannot be used to assess treatment outcome or the presence of active infection in this population.     

Analysis of FOXP3<Superscript>+</Superscript> regulatory T cell subpopulations in peripheral blood and tissue of patients with systemic lupus erythematosus

Regulatory T cells (Tregs) are critical mediators of immune tolerance, yet their involvement in the autoimmune disease systemic lupus erythematosus (SLE) is incompletely understood. We analyzed CD4⁺ T cell subpopulations with Treg-related phenotypes and their association with disease activity in peripheral blood (PB) and tissues of patients with SLE. In detail, we quantified subpopulations regarding CD25, FOXP3, CD62L, CCR6, CD27, CD45RA, and CD45RO expression in PB from 31 patients with SLE divided into two disease activity groups and 32 healthy controls using flow cytometry. CD4⁺ and FOXP3⁺ T cells in skin and kidney biopsies of patients with SLE were quantified by immunohistochemistry. CD4⁺CD25^+/++FOXP3⁺ and CD4⁺CD25⁺CD45RA^?/CD45RO⁺ T cell frequencies were significantly higher in PB from patients with active compared to inactive SLE. The fraction of CD4⁺CD25⁺⁺FOXP3⁺ Tregs and CD4⁺CD25⁺CD45RA⁺/CD45RO^? naïve Tregs was not significantly different between these groups. CD4⁺CD25⁺⁺ Tregs from active SLE patients comprised significantly less CD27⁺ cells and more CCR6⁺ cells compared to patients with inactive SLE. The percentage of CD4⁺FOXP3⁺ T cells among inflammatory infiltrates in skin and kidney biopsies of SLE patients was not different from other inflammatory skin/kidney diseases. In conclusion, although CD4⁺FOXP3⁺ T cell frequencies in the inflamed tissues of SLE patients were comparable to other inflammatory diseases, distinct T cell subpopulations appeared misbalanced in PB of patients with active SLE. Here, cells phenotypically resembling activated T cells, but not Tregs, were increased compared to patients with inactive SLE. Within Tregs of patients with active SLE, markers related to Treg function and homing were altered.     

Membrane potential modulation of ionomycin-stimulated Ca<Superscript>2+</Superscript> entry via Ca<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange and SOC in rat submandibular acinar cells

Ionomycin (IM) at 5 μM mediates the Ca²⁺/H⁺ exchange, while IM at 1 μM activates the store-operated Ca²⁺ entry channels (SOCs). In this study, the effects of depolarization on both pathways were examined in rat submandibular acinar cells by increasing extracellular K⁺ concentration ([K⁺]_o). IM (5 μM, the Ca²⁺/H⁺ exchange) increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) to an extremely high value at 151 mM [K⁺]_o. However, with increasing [K⁺]_o, the rates of Ca²⁺ entry decreased in a linear relationship. The reversal potential (E _rev) for the Ca²⁺/H⁺ exchange was +93 mV, suggesting that IM (5 μM) exchanges 1 Ca²⁺ for 1 H⁺. Thus, depolarization decreases the Ca²⁺ influx via the Ca²⁺/H⁺ exchange because of its electrogenicity (1 Ca²⁺ for 1 H⁺). On the other hand, IM (1 μM, the SOCs) abolished an increase in [Ca²⁺]_i at 151 mM [K⁺]_o. With increasing [K⁺]_o, the rate of Ca²⁺ entry immediately decreased linearly. The E _rev for the SOC was +3.7 mV, suggesting that the SOCs are nonselective cation channels and less selective for Ca²⁺ over Na⁺ (P _Ca/P _Na = 8.2). Moreover, an increase in extracellular Ca²⁺ concentration (20 mM) enhanced the Ca²⁺ entry via the SOCs at 151 mM [K⁺]_o, suggesting depolarization does not inhibit the SOCs and decreases the driving force for the Ca²⁺ entry. This suggests that membrane potential changes induced by a secretory stimulation finely regulate the [Ca²⁺]_i via the SOCs in rat submandibular acinar cells. In conclusion, IM increases [Ca²⁺]_i via two pathways depending on its concentration, the exchange of 1 Ca²⁺ for 1 H⁺ at 5 μM and the SOCs at 1 μM.     

Lenalidomide potentiates CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>Treg-related suppression of lymphoma B-cell proliferation

We have previously found that ex vivo expanded human CD4⁺CD25⁺Treg cells suppress proliferation of lymphoma B-cell lines. Here we demonstrate that the immunomodulatory drug lenalidomide potentiates suppression of lymphoma B-cell proliferation by freshly isolated CD4⁺CD25⁺Tregs, as well as suppression by Tregs expanded polyclonally in the presence of rapamycin from CD4⁺CD25⁺T cells or CD4⁺CD25⁺CD127^loT cells. The regulation of lymphoma cell proliferation by Tregs pre-expanded with “third-party” allogeneic MoDCs in the presence of rapamycin was also potentiated by lenalidomide. Lenalidomide contributed to the suppression exerted by Tregs despite concomitant downregulation of Treg proliferation. Lenalidomide did not reduce the suppression of conventional T cells by expanded Tregs. The exposure of polyclonally expanded Tregs to lenalidomide did not significantly alter their phenotype. There was no uniform pattern of lenalidomide effect on Treg-mediated regulation of lymphoma B cells freshly isolated from patients. Freshly isolated lymphoma cells activated with multimeric CD40L and IL-4 to support their survival in vitro varied in their sensitivity to lenalidomide, and the regulatory effect of Tregs on such lymphoma cells ranged from suppression to help in individual patients. Lenalidomide potentiated or attenuated Treg effects on the survival of freshly isolated lymphoma cells. A combination of lenalidomide treatment with adoptive transfer of CD4⁺CD25⁺Tregs or CD4⁺CD25⁺CD127^loTregs expanded ex vivo could be used to suppress proliferation of residual lymphoma in select patients with lymphoma responsive to the regulation by Tregs and sensitive to lenalidomide.