Fos-like immunoreactivity induced by intraplantar injection of endotoxin and its reduction by morphine

C-Fos-like immunoreactivity (FLI) in the central nervous system, has been associated with the processing of nociceptive information in acute and chronic pain animal models. The aim of this study was to investigate whether intraplantar (i.pl.) injections of endotoxin (ET, 1.25 μg) can induce FLI in the lumbar spinal cord of rats and to assess the effects of morphine injection on c-fos expression. FLI was studied in various groups of rats at 2, 3, 4, 6, 9 and 24 h following ET injections. Labeled neurons were mainly detected in the lumbar segments ipsilateral to the ET-injected leg, with a major peak (71.01±4.79 positive neurons) at 4 h and a second peak (29.87±5.97 positive neurons) at 9 h followed by a recovery to the baseline at 24 h after ET injections. Within the laminae, the majority of positive neurons was observed at 2–3 h in laminae I and II and in deep laminae (V and VI mainly) starting at 4 h after ET injections. Rostrocaudally, labeled neurons were observed initially in L₄–L₅ segments (2–3 h post-ET) after which they extended to L₂–L₆ segments at 4 h after ET. Morphine injections either i.p. (1 or 2 mg/kg) or i.pl. (50 μg) significantly reduced ET-induced hyperalgesia and simultaneously the FLI. The maximum effect was observed on labeled neurons in the deep laminae (V and VI mainly). We conclude that local injections of ET can induce FLI in the lumbar spinal cord with a temporal and spatial patterns comparable to the described hyperalgesia, and that both FLI and hyperalgesia are reduced by morphine in a dose-dependent manner with a maximal effect shown by the local i.pl. morphine injections.     

Supraspinal modulation of nociception in awake rats by stimulation of the dorsal column nuclei

Rats with bilateral dorsal column (DC) lesions and chronic placement of stimulating electrodes on DC nuclei (DCN) and sham-operated rats were studied using the tail immersion test for phasic pain and the formalin test for tonic pain. DCN stimulation produced a clear inhibitory effect, through a supraspinal loop, in both phasic and tonic pain in awake rats.     

Supraspinal inhibition of trigeminal input into subnucleus caudalis by dorsal column stimulation

In decorticate-decerebellate cats with cervical spinal cuts which isolated the dorsal columns (DCs), conditioning volleys travelling in the DCs inhibited activities of trigemenal caudalis neurons. This inhibition was most likely mediated through a brainstem loop and was particularly effective on nociceptive-driven neurons.     

Interactions of inputs from dorsal columns and ventral tracts with visual inputs on single neurons of cat superior colliculus

In cats with upper spinal lesions disconnecting the dorsal column (DC) from the spinocervicolemniscal and ventral tract (VT) systems, single unit responses in the superior colliculus could be evoked by DC and VT stimulation. Excitatory convergence, as well as facilitatory and inhibitory interactions, were observed between DC, VT, visual and auditory inputs.     

Plasmacytoid dendritic cells (PDC) are the major DC subset innately producing cytokines in human lymph nodes

Plasmacytoid dendritic cells (PDC) constitute a distinct subset of DC found in human peripheral lymph nodes (LN), but little is known about their function. Cell suspensions were prepared from tumor draining LN (n=20) and control LN (n=11) of women undergoing surgical resection for primary breast cancer and elective surgery for benign conditions, respectively. Using four-color flow cytometry, human leukocyte antigen-DR+ DC subsets were identified phenotypically. The proportions and numbers of cells innately producing interleukin (IL)-4, IL-10, IL-12, and interferon-gamma (IFN-gamma) were also measured from intracellular accumulation of cytokine after blocking with monensin. All flow cytometry data were collected without compensation and were compensated off-line using the Winlist algorithm (Verity software). This package also provided the subtraction program to calculate percentage positive cells and intensity of staining. PDC (CD11c-, CD123+) expressed more cytokines than did myeloid DC (CD11c+) or CD1a+ putative "migratory" DC (P<0.001). LN PDC from patients with a good prognosis (px; n=11) demonstrated a relative increase in IL-12 and IFN-gamma expression (median IL-10:IL-12 ratio=0.78 and median IL-4:IFN-gamma ratio=0.7), and PDC from LN draining poor px cancer (n=9) showed a relative increase in IL-10 and IL-4 expression (median IL-10:IL-12 ratio=1.31 and median IL-4:IFN-gamma ratio=2.6). The difference in IL-4:IFN-gamma expression between good and poor px cancer groups was significant (P<0.05). Thus, PDC innately producing cytokines were identified in cell suspensions from human LN, and the character of PDC cytokine secretion may differ between two breast cancer prognostic groups. We speculate that a shift towards PDC IL-10 and IL-4 expression could promote tumor tolerance in LN draining poor px breast cancer.     

It's About Everything: Validation and Optimization of 96-Well Tray Flow Cytometry Crossmatch

Flow cytometric crossmatch (FCXM) is widely used in many centers as part of pretransplant risk assessment to detect donor-specific anti-HLA antibodies. The limited number of crossmatches that can be performed during on-call work-up for deceased donors within reasonable time remains the main obstacle to accommodating the majority of highly sensitized listed patients to be tested by the standard tube FCXM method. This limitation often directs the organs to nonsensitized patients and deprives highly sensitized patients who could be compatible if their sera were included in the crossmatch test.The goal of this study is to optimize a 96-well tray FCXM protocol that allows more sera to be crossmatched without prolonging the overall procedural time while maintaining quality and sensitivity of the assay. The new method was validated against use of the standard tube method and included total of 63 crossmatches performed simultaneously by both methods using 20 donors' cells with patients' sera, pooled positive controls tested on different dilutions, and commercial negative control.In the new protocol we modify various assay parameters including tube platform, incubation time, amount of reagent antibody cocktail, and cell volumes. An overall concordance of 98% was achieved with the protocols with slight improvement in sensitivity (2 negative B-cell reactions converted to positive in presence of weak donor-specific antibodies and mild T-cell reactivity could be picked up at 1:80 diluted positive control by the tray method only). The median channel fluorescence values of the 2 methods were essentially equivalent for both T and B crossmatches (r² of 0.98 and 0.97, respectively). In conclusion, 96-well tray assay has the potential to increase the probability of highly sensitized patients receiving transplants by allowing increased number of crossmatches to be performed with significant reduction in turnaround time and assay cost. Furthermore, the enhanced sensitivity of the assay will provide more accurate information about sensitization status and strength of donor-specific antibodies to treating physicians, allowing them to choose the best therapeutic option and to provide better patient care.     

Cannabinoid 2 receptor induction by IL-12 and its potential as a therapeutic target for the treatment of anaplastic thyroid carcinoma

Anaplastic thyroid carcinoma is the most aggressive type of thyroid malignancies. Previously, we demonstrated that tumorigenicity of anaplastic thyroid carcinoma cell line ARO was significantly reduced following interleukin (IL)-12 gene transfer. We suspected that tumor target structure in ARO/IL-12 cells might be changed and such a change may make them more susceptible to be killed through mechanisms apart from natural killer-dependent pathway. To identify genes involved, we examined gene expression profile of ARO and ARO/IL-12 by microarray analysis of 3757 genes. The most highly expressed gene was cannabinoid receptor 2 (CB2), which was expressed eightfold higher in ARO/IL-12 cells than ARO cells. CB2 agonist JWH133 and mixed CB1/CB2 agonist WIN-55,212-2 could induce significantly higher rate of apoptosis in ARO/IL-12 than ARO cells. Similar results were obtained when ARO cells were transfected with CB2 transgene (ARO/CB2). A considerable regression of thyroid tumors generated by inoculation of ARO/CB2 cells was observed in nude mice following local administration of JWH133. We also demonstrated significant increase in the induction of apoptosis in ARO/IL12 and ARO/CB2 cells following incubation with 15 nM paclitaxel, indicating that tumor cells were sensitized to chemotherapy. These data suggest that CB2 overexpression may contribute to the regression of human anaplastic thyroid tumor in nude mice following IL-12 gene transfer. Given that cannabinoids have shown antitumor effects in many types of cancer models, CB2 may be a viable therapeutic target for the treatment of anaplastic thyroid carcinoma.     

Alkhurma hemorrhagic fever in humans, Najran, Saudi Arabia

Alkhurma virus is a flavivirus, discovered in 1994 in a person who died of hemorrhagic fever after slaughtering a sheep from the city of Alkhurma, Saudi Arabia. Since then, several cases of Alkhurma hemorrhagic fever (ALKHF), with fatality rates up to 25%, have been documented. From January 1, 2006, through April 1, 2009, active disease surveillance and serologic testing of household contacts identified ALKHF in 28 persons in Najran, Saudi Arabia. For epidemiologic comparison, serologic testing of household and neighborhood controls identified 65 serologically negative persons. Among ALKHF patients, 11 were hospitalized and 17 had subclinical infection. Univariate analysis indicated that the following were associated with Alkhurma virus infection: contact with domestic animals, feeding and slaughtering animals, handling raw meat products, drinking unpasteurized milk, and being bitten by a tick. After multivariate modeling, the following associations remained significant: animal contact, neighboring farms, and tick bites.