Cooperativity Between Selectins and β2-Integrins Define Neutrophil Capture and Stable Adhesion in Shear Flow

A cooperative, sequential process of molecular recognition governs leukocyte capture, rolling, and arrest on inflamed endothelium. Flowing neutrophils are captured via heterotypic adhesive interactions mediated by endothelial E-selectin, whereas homotypic interactions between neutrophils are mediated by L-selectin. To elucidate how each selectin facilitates the transition to CD18-mediated stable adhesion, E-selectin and L-selectin were expressed at defined site density in a murine pre-B-cell line. Direct observation of two-body collisions revealed that 30% of neutrophil interactions with E-selectin transfectants formed doublets at low shear rate G = 14 s(-1) whereas a threshold shear rate 14 s(-1) < or = G < or = 10 s(-1) was necessary for L-selectin adhesion. Adhesion via L-selectin resisted rupture at high shear stress, while E-selectin tethered doublets remained intact longer once formed. Moreover, higher expression of L-selectin (1100 sites/microm2) than that of E-selectin (220 sites/microm2) was required for comparable heterotypic adhesion efficiency. With a threefold rise in active CD18 upregulated on chemotactically stimulated neutrophils, homotypic adhesion efficiency increased 10-fold compared to less than 5-fold for heterotypic adhesion to selectin transfectants. Co-expression of E-selectin and ICAM-1 boosted adhesion efficiency threefold more than either receptor alone over the range of active CD18 expression. These data are the first to quantify adhesion efficiency mediated by selectin tethering and conformational activation of beta2-integrin in neutrophils in shear flow.     

Leukemic recombinations involving heterochromatin in myeloproliferative disorders with t(1;9)

The unbalanced t(1;9) is a rare, recurrent rearrangement in polycythemia vera (PV) resulting in trisomy of both 1q and 9p arms, whereas a balanced t(1;9)(q12;q12), to our knowledge, has never been reported before. We studied two patients with PV and one with idiopathic myelofibrosis bearing an unbalanced t(1;9) and one patient with essential thrombocythemia with a balanced t(1;9). In all cases fluorescence in situ hybridization showed that the breakpoints were located within the satellite II family of heterochromatin of chromosome 1 and the satellite III of chromosome 9. Heterochromatin breakage and reunion produce the unbalanced t(1;9) and may contribute to a gene dosage effect due to gains of 1q and 9p. Case 4 with the balanced t(1;9), however, suggests that translocation of heterochromatin close to critical genes could interfere with their function. The molecular event underlying juxtaposition of satellite II of chromosome 1 and the satellite III of chromosome 9 remains to be elucidated.     

Membrane androgen receptor activation induces apoptotic regression of human prostate cancer cells in vitro and in vivo

Nongenomic androgen actions imply mechanisms different from the classical intracellular androgen receptor (iAR) activation. We have recently reported the identification of a membrane androgen receptor (mAR) on LNCaP human prostate cancer cells, mediating testosterone signal transduction within minutes. In the present study we provide evidence that activation of mAR by nonpermeable, BSA-coupled testosterone results in 1) inhibition of LNCaP cell growth (with a 50% inhibitory concentration of 5.08 nM, similar to the affinity of testosterone for membrane sites); 2) induction in LNCaP cells of both apoptosis and the proapoptotic Fas protein; and 3) a significant decrease in migration, adhesion, and invasion of iAR-negative DU145 human prostate cancer cells. These actions persisted in the presence of antiandrogen flutamide or after decreasing the content of iAR in LNCaP cells by iAR antisense oligonucleotides. Testosterone-BSA was also effective in inducing apoptosis of DU145 human prostate cancer cells, negative for iAR, but expressing mAR sites. In LNCaP cell-inoculated nude mice, treatment with testosterone-BSA (4.8 mg/kg body weight) for 1 month resulted in a 60% reduction of tumor size compared with that in control animals receiving only BSA, an effect that was not affected by the antiandrogen flutamide. Our findings suggest that activators of mAR may represent a new class of antitumoral agents of prostate cancer.     

Q fever in children in Greece

The aim of this study was to investigate the incidence, epidemiology, and clinical manifestations of Q fever among hospitalized children in Greece. During a two-year period, 1,200 children with various clinical manifestations were prospectively tested for Coxiella burnetii infection by indirect immunofluorescence. Acute Q fever was diagnosed in eight (0.67%) patients. No chronic case of infection was detected. Multivariate analysis showed that children 11-14 years old and children reporting consumption of cheese from rural areas were at increased risk for this illness. Clinical manifestations of acute Q fever were pneumonia (two patients), meningitis (two), prolonged fever (two), hepatitis (one), and hemolytic-uremic syndrome (one). Q fever accounted for 2.9% of the cases with prolonged fever, 1.2% of the cases of meningitis, and 0.5% of the cases of pneumonia. Fever and headache were the most common symptoms at presentation. Our study indicates that Q fever is a rare cause of hospitalization during childhood.     

Recovery, innervation profile, and contractile properties of reinnervating fast muscles following postnatal nerve crush and administration of L-Dopa

Muscle and peripheral nerve development is clearly dependent on their interaction during early postnatal life. Furthermore, muscle or peripheral nerve activity plays a crucial role in the maturation of the neuromuscular system. In this study, the possible involvement of spinal catecholamines in fast muscle recovery after nerve crush is investigated. Sciatic nerve crush was performed on the fourth to fifth postnatal day. Following that, L-Dopa was administered daily [150 mg/kg body weight (BW)] i.p., until the 21st day after birth. L-Dopa-treated and control groups were then examined electrophysiologically for the contractile properties of extensor digitorum longus (EDL) muscles. Two experimental groups were included in this study: (i) rats whose sciatic nerve was crushed and were treated with L-Dopa and (ii) rats whose sciatic nerve was crushed and were not treated with L-Dopa. The number of motoneurones for both groups was estimated by HRP retrograde labelling. The results showed that the operated L-Dopa-treated EDL muscles of the rats exhibited limited atrophy, slighter impairment of maximal tetanic tension, lesser resistance to fatigue, and polyneuronal innervation than the controls. The number of motoneurones was the same for the operated muscles in both groups of animals and was within the normal ranges. Our findings suggest that catecholamines of locomotion during the early stages of development may have a beneficial effect on fast muscle recovery following nerve crush. The action of L-Dopa is attributed to noradrenaline, which acts through descending spinal noradrenergic pathways, possibly via a(2)-adrenergic receptors at the spinal level.     

Hypoxia-ischemia affects erythropoietin and erythropoietin receptor expression pattern in the neonatal rat brain

Erythropoietin (EPO), known for its role in erythroid differentiation, has been suggested to have non-hematopoietic functions in the brain, especially during development. In the present study, we investigated the expression of erythropoietin and erythropoietin receptor (EPOR) in the developing rat brain following hypoxia-ischemia. Seven-day-old rats underwent unilateral, permanent carotid artery ligation followed by 1 h of hypoxia, and their brains were examined immediately, 24 h or 4 days after hypoxia-ischemia. RT-PCR and Western blot analysis revealed that hypoxia-ischemia only marginally affected EPO expression. Immunohistochemical study of brains 4 days after hypoxia showed that 60 min of hypoxia (resulting in cortical infarction and severe neuronal loss in other regions) led to the increased EPO immunoreactivity, especially in the boundaries of the damaged cerebral cortex, associated with astrocytosis. In contrast, EPOR was dramatically upregulated within 24 h after hypoxia-ischemia. These results suggest that there is a rapid response of EPOR to the hypoxic-ischemic stimulus, which seems to precede that of EPO, leading to the hypothesis that the EPO/EPOR system is implicated in the processes of neuroprotection from hypoxia-ischemia.     

Changes of gastric emptying rate and gastrin levels are early indicators of autonomic neuropathy in type II diabetic patients

The authors investigated the effect of a balanced meal on gastric emptying rate and gastrin plasma concentrations in patients with type II diabetes and autonomic neuropathy, in diabetic patients without autonomic neuropathy, and in healthy subjects (controls). Before food the gastrin plasma concentrations were higher in patients with diabetes with autonomic neuropathy. After food, gastric emptying rate was slower in patients with diabetes with autonomic neuropathy, whereas gastrin plasma concentrations increased in 30 minutes in all groups but to a greater extent in patients with diabetes with autonomic neuropathy. Sixty minutes after food, there was a significant decrease in gastrin plasma concentrations in patients with diabetes with autonomic neuropathy, compared with the other two groups. These data suggest that in patients with type II diabetes with autonomic neuropathy, food causes slower gastric emptying and different plasma gastrin level responses from those in patients with type II diabetes without autonomic neuropathy and controls. There are therefore differences in the responses to food ingestion between these groups because of vagal denervation induced by autonomic neuropathy. These tests should be reserved for patients with symptoms suggestive of disturbed gastric emptying, or for patients with autonomic neuropathy without symptoms of gastroparesis.     

Effects of homo-aza-steroids on acute non-lymphocytic leukaemia cell proliferation in vitro

Summary. Homo-aza-steroids (modified steroid molecules) in their esterified forms have been used extensively as carrier molecules of alkylating agents against several neoplastic malignancies in vivo and in vitro. We studied the effects of two homo-aza-steroid carrier molecules alone, namely 3β-hydroxy-13α-amino-13,17-seco-5α-androstan-17-oic-13, 17-lactam (compound 1) and 13α-amino-13,17-seco-1,3,5-estratrien-17-oic-13,17-lactam (compound 2), on human acute non-lymphocytic leukaemia cell proliferation in vitro.
We used peripheral blood samples from 27 untreated ANLL patients (eight M1, four M2, two M3, six M4, three M5a, two M5b and two M6, according to FAB criteria). Proliferative activity was estimated by using thymidine uptake and the percentage of cells in metaphase in 24, 48 and 72 h of culture. Exposure of human leukaemic blasts with either of the two compounds resulted in enhanced cell proliferation in M1, M2, M4, M6 and M5a (only by compound 2) cases, whereas there was no significant effect in the M3 and M5b cases.
Our results indicate that the two compounds tested exhibit stimulatory effect on cell proliferation, particularly in blast cells possessing a relatively smaller degree of differentiation (Ml and M6 cases exhibiting CD34 and CD7). Further research is needed to study the cell growth effect and the therapeutic potential of these steroid molecules in human blood malignancies in vitro and in vivo.     

WT1-specific T cell receptor gene therapy: improving TCR function in transduced T cells

Adoptive transfer of antigen-specific T lymphocytes is an attractive form of immunotherapy for haematological malignancies and cancer. The difficulty of isolating antigen-specific T lymphocytes for individual patients limits the more widespread use of adoptive T cell therapy. The demonstration that cloned T cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T cell therapy. The first trial in humans demonstrated that TCR gene-modified T cells persisted for an extended time period and reduced tumor burden in some patients. The WT1 protein is an attractive target for immunotherapy of leukemia and solid cancer since elevated expression has been demonstrated in AML, CML, MDS and in breast, colon and ovarian cancer. In the past, we have isolated high avidity CTL specific for a WT1-derived peptide presented by HLA-A2 and cloned the TCR alpha and beta genes of a WT1-specific CTL line. The genes were inserted into retroviral vectors for transduction of human peripheral blood T lymphocytes of leukemia patients and normal donors. The treatment of leukemia-bearing NOD/SCID mice with T cells transduced with the WT1-specific TCR eliminated leukemia cells in the bone marrow of most mice, while treatment with T cells transduced with a TCR of irrelevant specificity did not diminish the leukemia burden. In order to improve the safety and efficacy of TCR gene therapy, we have developed lentiviral TCR gene transfer. In addition, we employed strategies to enhance TCR expression while avoiding TCR mis-pairing. It may be possible to generate dominant TCR constructs that can suppress the expression of the endogenous TCR on the surface of transduced T cells. The development of new TCR gene constructs holds great promise for the safe and effective delivery of TCR gene therapy for the treatment of malignancies.