A simple and fast way to count heterophils in chemotaxis

Heterophils were labeled with fluorescein isothiocyanate and then placed in a modified Boyden chamber to determine their response to chemoattractants. After incubation, the cells that had migrated into the membrane were examined under an epifluorescent microscope, and the image was captured by a charge-coupled digital camera and a frame grabber and saved. The saved images were then counted by NIH Image (a public domain software). These modifications greatly speed up the counting of cells and relieve the eyestrain suffered among workers in the chemotaxis field.     

Radiological features of focal nodular hyperplasia of the liver in children

BACKGROUND: Focal nodular hyperplasia (FNH) is an unusual hepatic tumour in children and should be distinguished from other hepatic lesions. OBJECTIVE: To describe the imaging characteristics of FNH in children. MATERIALS AND METHODS: We examined five patients (three boys and two girls, mean age 9.4 years) with pathologically confirmed FNH. The diagnosis was obtained by tumour resection (n = 4) and percutaneous needle biopsy (n = 1). One patient with multiple FNHs showed recurrent lesions after tumour resection. All patients were studied with US (including colour and power Doppler US [n = 3]) and CT. Dynamic enhanced CT scans were available in three patients. MRI (n = 2) or coeliac angiography (n = 1) was performed in three patients. RESULTS: Seven of eight FNH lesions in five patients were demonstrated by imaging. The average size of the lesions was 6.5 cm. Six lesions detected on US showed variable echogenicity with a central hyperechoic scar (n = 2). On Doppler examination, central or peripheral hypervascular areas were seen (n = 3). Six lesions detected on contrast-enhanced CT showed high attenuation (n = 4) or iso-attenuation (n = 2). On early phase scans, all the lesions (n = 3) showed high attenuation. Irregular linear or ovoid central scars were detected in two patients on CT. MR demonstrated three lesions in two patients, one of which had not been detected by US or CT. A central low signal intensity scar (n = 1) was seen on T2-weighted MRI. Coeliac angiography performed in one patient showed a hypervascular mass with homogeneous staining. CONCLUSION: FNH in children shows a wide spectrum of imaging findings on various radiological examinations and the typical central scar was not always seen on imaging studies. Dynamic enhanced CT obtained in the early phase and colour Doppler studies may be helpful in the diagnosis of FNH by allowing characterisation of tumour vascularity. FNH should be included in the differential diagnosis of liver mass in children.     

Cytosolic enzymes with a mitochondrial ancestry from the anaerobic chytrid Piromyces sp. E2

The anaerobic chytrid Piromyces sp. E2 lacks mitochondria, but contains hydrogen-producing organelles, the hydrogenosomes. We are interested in how the adaptation to anaerobiosis influenced enzyme compartmentalization in this organism. Random sequencing of a cDNA library from Piromyces sp. E2 resulted in the isolation of cDNAs encoding malate dehydrogenase, aconitase and acetohydroxyacid reductoisomerase. Phylogenetic analysis of the deduced amino acid sequences revealed that they are closely related to their mitochondrial homologues from aerobic eukaryotes. However, the deduced sequences lack N-terminal extensions, which function as mitochondrial leader sequences in the corresponding mitochondrial enzymes from aerobic eukaryotes. Subcellular fractionation and enzyme assays confirmed that the corresponding enzymes are located in the cytosol. As anaerobic chytrids evolved from aerobic, mitochondria-bearing ancestors, we suggest that, in the course of the adaptation from an aerobic to an anaerobic lifestyle, mitochondrial enzymes were retargeted to the cytosol with the concomitant loss of their N-terminal leader sequences.     

Digital image analysis of hematopoietic colonies in vitro

A system for automatic analysis of in vitro hematopoietic colonies is described and evaluated. With the standard resolution provided by video cameras, the improvement in visualization obtained using features other than size and darkness when classifying potential colonies appears to be limited. We confirmed this by comparing results obtained with the test system with those obtained with a commercial one. However, for some applications it may be useful to supplement the system with specific methods, e.g., to separate merged colonies. Digital image analyses provide new possibilities, for instance of measuring the total cellularity of the dish or analyzing colonies according to the size and cell density of each colony. Examples provided are time course studies of colony development, cellularity feedback effects on colony sizes, and bell-shaped dose-response curves for the growth stimulation obtained by certain conditioned media on a subpopulation of progenitor cells that gives rise to large colonies.     

Infants' detection of increments in low- and high-frequency noise

A visually reinforced operant paradigm was employed to examine the relationship between the difference limen (DL) for intensity and level of the standard during infancy. In Experiment 1, 7-month-old infants and adults detected increments in continuous noise presented via headphones at each of four levels ranging from 28 to 58 dB SPL. Noise stimuli were 2-octave bands centered at either 400 or 4000 Hz, and increments were 10 and 100 msec in duration. Infants' DLs were significantly larger than those of adult subjects and significantly larger for low- than for high-frequency stimuli. For the high-frequency noise band, infants' DLs were generally consistent with Weber's law, remaining essentially constant for standards higher than 28 dB SPL (3 dB SL) for 100-msec increments and 38 dB SPL (13 dB SL) for 10-msec increments. For low-frequency noise, infants' absolute thresholds were exceptionally high, and sensation levels of the standards were too low to adequately describe the relationship. In Experiment 2, 7-month-old infants detected 10- and 100-msec increments in 400-Hz noise stimuli presented in sound field. Infants' low-frequency DLs were large at low intensities and decreased with increases in level of the standard up to at least 30 dB SL. For both low- and high-frequency noise, the difference between DLs for 10- and 100-msec increments tended to be large at low levels of the standard and to decrease at higher levels. These results suggest that the relationship between the DL and level of the standard varies with both stimulus frequency and duration during infancy. However, stimulus-dependent immaturities in increment detection may be most evident at levels within approximately 30 dB of absolute threshold.     

5-Aminolevulinic acid induced endogenous porphyrin fluorescence in 9L and C6 brain tumours and in the normal rat brain

A new approach in photodynamic therapy is the use of endogenous porphyrins for sensitisation of tumours to light. The induction of endogenous porphyrins after intravenous injection of 5-aminolevulinic acid (ALA, 200 mg kg-1) was studied in 23 rats, bearing intracranial 9L or C6 tumours. After 0, 2, 4, 6, 8, and 22 hours the rats were sacrificed and the fluorescence distribution of endogenous porphyrins was studied in brain tissue sections with a standard fluorescence microscope and a confocal laser scanning microscope. The role of blood-brain barrier disruption on porphyrin production was studied in 2 rats with a cryo-lesion of the cortex. Additionally, 9L and C6 tumour cell cultures were incubated with ALA for 8 hours in vitro. Fluorescence was measured with a fluorescence spectrophotometer in cell cultures and in the brain sections. Porphyrins were detected in vitro in the tumour cells from 2 hours onwards and ex vivo in the tumour sections mainly from 2 to 8 hours, by 22 hours porphyrin fluorescence had almost disappeared. The contralateral brain showed low fluorescence levels between 2 and 6 hours after ALA administration. At the site of the cryo-lesions low fluorescence was measured 6 hours after ALA administration. The 9L tumours fluoresced homogeneously, with a sharp demarcation towards normal brain tissue. Fluorescence in the C6 tumours was patchy, with a poorly fluorescing edge. In both tumour models fluorescence was also detected in brain surrounding the tumour and sometimes in contralateral white matter and ventricle ependyma and pia mater. The slight increase of porphyrin fluorescence in the normal brain of tumour bearing rats, compared to the absence of this in rats without a tumour, was attributed to transport by bulk flow of porphyrins made in the tumours, and possibly also of circulating porphyrins or ALA leaking from the tumour vessels.     

Role of the TRAF binding site and NF-kappaB activation in Epstein-Barr virus latent membrane protein 1-induced cell gene expression

In this study, we investigated the induction of cellular gene expression by the Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1). Previously, LMP1 was shown to induce the expression of ICAM-1, LFA-3, CD40, and EBI3 in EBV-negative Burkitt lymphoma (BL) cells and of the epidermal growth factor receptor (EGF-R) in epithelial cells. We now show that LMP1 expression also increased Fas and tumor necrosis factor receptor-associated factor 1 (TRAF1) in BL cells. LMP1 mediates NF-kappaB activation via two independent domains located in its C-terminal cytoplasmic tail, a TRAF-interacting site that associates with TRAF1, -2, -3, and -5 through a PXQXT/S core motif and a TRADD-interacting site. In EBV-transformed B cells or transiently transfected BL cells, significant amounts of TRAF1, -2, -3, and -5 are associated with LMP1. In epithelial cells, very little TRAF1 is expressed, and only TRAF2, -3, and -5, are significantly complexed with LMP1. The importance of TRAF binding to the PXQXT/S motif in LMP1-mediated gene induction was studied by using an LMP1 mutant that contains alanine point mutations in this motif and fails to associate with TRAFs. This mutant, LMP1(P204A/Q206A), induced 60% of wild-type LMP1 NF-kappaB activation and had approximately 60% of wild-type LMP1 effect on Fas, ICAM-1, CD40, and LFA-3 induction. In contrast, LMP1(P204A/Q206A) was substantially more impaired in TRAF1, EBI3, and EGF-R induction. Thus, TRAF binding to the PXQXT/S motif has a nonessential role in up-regulating Fas, ICAM-1, CD40, and LFA-3 expression and a critical role in up-regulating TRAF1, EBI3, and EGF-R expression. Further, D1 LMP1, an LMP1 mutant that does not aggregate failed to induce TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 expression confirming the essential role for aggregation in LMP1 signaling. Overexpression of a dominant form of IkappaBalpha blocked LMP1-mediated TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 up-regulation, indicating that NF-kappaB is an important component of LMP1-mediated gene induction from both the TRAF- and TRADD-interacting sites.