Receptive field scatter,topography and map variability in different layers of the hindpaw representation of rat somatosensory cortex

We recorded neurons extracellularly in layers II/III, IV, and V of the hindpaw representation of primary somatosensory cortex in anesthetized rats and studied laminar features of receptive fields (RFs) and representational maps. On average, RFs were smallest in layer IV and largest in layer V; however, for individual penetrations we found substantial deviations from this rule. Within the hindpaw representation, a distinct rostrocaudal gradient of RF size was present in all layers. While layer V RFs were generally largest independent of this gradient, layer IV RFs recorded caudally representing the proximal portions of the paw were larger than layer II/III RFs recorded rostrally representing the digits. The individual scatter of the locations of RFs across laminar groups was in the range of several millimeters, corresponding to about 25% of the average RF diameter. The cutaneous representations of the hindpaw in extragranular layers were confined to the areal extent defined by responsive sites in layer IV. Comparison between RFs determined quantitatively and by handplotting showed a reliable correspondence. Repeated measurements of RFs revealed spontaneous fluctuations of RF size of no more than 5% of the initial condition over an observation period of several hours. The topography and variability of cortical maps of the hindpaw representation were studied with a quantitative interpolation method taking into account the geometric centers of RFs and the corresponding cortical recording sites. On average, the overall topography in terms of preservation of neighborhood relations was present in all layers, although some individual maps showed severe distortions of topography. Factors contributing to map variability were overall position of the representation on the cortical surface, internal topography and spatial extent. Interindividual variability of map layout was always highest in the digit representations. Local topographic orderliness was lowest in layer V, but comparable in layers II/III and IV. Within layer IV, the lowest orderliness was observed in the digit representations. Our data emphasize a substantial variability of RF size, overlap and position across layers and within layers. At the level of representational maps, we found a similar degree of variability that often co-varied across layers, with little evidence for significant layer specificity. Laminar differences are likely to arise from the specific input-output pattern, layer-specific cell types and the connectivity between different layers. Our findings emphasizing similarities in the variability across layers support the notion of tightly coupled columnar interactions between different layers.     

Silver‐Russell patients showing a broad range of ICR1 and ICR2 hypomethylation in different tissues

Begemann M, Spengler S, Kanber D, Haake A, Baudis M, Leisten I, Binder G, Markus S, Rupprecht T, Segerer H, Fricke‐Otto S, Mühlenberg R, Siebert R, Buiting K, Eggermann T. Silver‐Russell patients showing a broad range of ICR1 and ICR2 hypomethylation in different tissues. In all known congenital imprinting disorders an association with aberrant methylation or mutations at specific loci was well established. However, several patients with transient neonatal diabetes mellitus (TNDM), Silver‐Russell syndrome (SRS) and Beckwith‐Wiedemann syndrome (BWS) exhibiting multilocus hypomethylation (MLH) have meanwhile been described. Whereas TNDM patients with MLH show clinical symptoms different from carriers with isolated 6q24 aberrations, MLH carriers diagnosed as BWS or SRS present only the syndrome‐specific features. Interestingly, SRS and BWS patients with nearly identical MLH patterns in leukocytes have been identified. We now report on the molecular findings in DNA in three SRS patients with hypomethylation of both 11p15 imprinted control regions (ICRs) in leukocytes. One patient was a monozygotic (MZ) twin, another was a triplet. While the hypomethylation affected both oppositely imprinted 11p15 ICRs in leukocytes, in buccal swab DNA only the ICR1 hypomethylation was visible in two of our patients. In the non‐affected MZ twin of one of these patients, aberrant methylation was also present in leukocytes but neither in buccal swab DNA nor in skin fibroblasts. Despite mutation screening of several factors involved in establishment and maintenance of methylation marks including ZFP57, MBD3, DNMT1 and DNMT3L the molecular clue for the ICR1/ICR2 hypomethylation in our patients remained unclear. Furthermore, the reason for the development of the specific SRS phenotype is not obvious. In conclusion, our data reflect the broad range of epimutations in SRS and illustrate that an extensive molecular and clinical characterization of patients is necessary.     

IgA class antineutrophil cytoplasmic antibodies in primary sclerosing cholangitis and autoimmune hepatitis

Antineutrophil cytoplasmic antibodies (ANCA) of IgG class have been described at high prevalence in autoimmune hepatitis (AIH) and primary sclerosing cholangitis (PSC). Data on IgA class ANCA in these diseases are limited. The aim of this study was to determine the prevalence and fluorescence patterns of IgA class ANCA in AIH and PSC and to examine a relationship between the presence of IgA ANCA and clinical characteristics in these patients. Sera from 35 patients with PSC (21 with concomitant inflammatory bowel disease), 40 patients with AIH and 10 healthy controls were studied. ANCA were detected on ethanol-fixed neutrophils using an indirect immunofluorescence technique. ANCA of the IgA class were found in 20% of sera from patients with PSC and in 50% of AIH sera. The majority of AIH patients with IgA class ANCA showed a 'classical' perinuclear staining pattern, whereas the 'classical' and 'atypical' perinuclear fluorescence patterns were distributed equally in PSC. In sera containing IgG and IgA class ANCA simultaneously, IgG class ANCA showed an 'atypical' pANCA fluorescence pattern whereas IgA class ANCA produced a 'classical' perinuclear staining. The presence of IgA class ANCA was not associated with disease-specific clinical characteristics. IgA class ANCA are more frequently detected in sera of patients with AIH than PSC. The diversity of fluorescence patterns points to different target antigens of IgA class ANCA with distinct subcellular localizations.     

Enhancement of antibody dependent cellular cytotoxicity (ADCC) by combination of cytokines

Monoclonal antibodies (MAb) specific for tumor-associated antigens (TAA) can induce an immunological cellular attack of tumor cells by a process termed antibody dependent cellular cytotoxicity (ADCC). Cytokines may augment ADCC by direct activation of immune cells or by enhancement of TAA on tumor cells. Thus, we investigated whether ADCC by MAb 17-1A and BR55-2, which recognize TAA on colorectal tumor cells, can be augmented by 3-day incubation with different concentrations of IL-2, IL-4, IL-6, IL-12, IFN-alpha, IFN-gamma, GM-CSF, M-CSF, and TNF-alpha. ADCC was assessed by a new flowcytometric cytotoxicity assay (Flieger et al. Immunol Methods 1995; 180:1-13) using PKH-2 labeled HT29 cells as targets and PKH-26 labeled peripheral blood mononuclear cells from three healthy volunteers as effector cells. We found three reaction patterns with the cytokines tested: (a) cytokines, which increase ADCC (IL-2, IL-12, IFN-alpha, and IFN-gamma, which represent Thl cytokines); (b) cytokines with no effect (GM-CSF, M-CSF, and TNF-alpha); and (c) cytokines, which decrease ADCC (IL-4 and IL-6, which represent Th2 cytokines). Then, we tested cytokines that increase ADCC in combination with the other cytokines. We found that the combinations IL-2/IFN-alpha, IL-2/IFN-gamma, IL-2/IL-12, and IL-12/IFN-alpha potentiated ADCC. By contrast, IL-4 reduced the IL-2, IL-12, and IFN-alpha-induced ADCC. Since the Thl response, cooperation of monocytes and CD4 cells is involved, we plan to elucidate by magnetic cell sorting (MACS) separation techniques, which cells are involved in cytokine-induced ADCC. Our results may be useful for finding combinations of cytokines and MAb for the locoregional treatment of colorectal cancer.     

The role of alveolar macrophages in the pathogenesis of aspiration pneumonitis

RATIONALE: A robust TNFalpha response is seen following aspiration of food particles, while there is only a modest response to acid. OBJECTIVES: To examine the direct effects of acid and particulate components of gastric content on local and systemic macrophages. METHODS: Pathogen-free Long-Evans rats were injured with intratracheal instillation of normal saline (SHAM), low pH saline (ACID), small non-acidic particles (SNAP) or acidified particles (CASP). The alveolar (local) and the peritoneal (systemic) macrophages were harvested following the injury. MEASUREMENTS: We examined the phagocytic activity and TNFalpha release by the alveolar and peritoneal macrophages following in vivo and in vitro exposure to acid and/or food particles. TNFalpha release by macrophages was examined in response to E. coli lipopolysaccharide (LPS) stimulation. MAIN RESULTS: In rats injured with gastric particles, the number of the mononuclear cells was higher than those obtained from acid-injured animals. Both in vivo and in vitro exposure of the alveolar macrophages to SNAP resulted in increased production of TNFalpha within 8 hours. Transient exposure of the alveolar macrophages to a low pH environment suppressed LPS-induced production of this cytokine. Additionally, the phagocytic activity of the alveolar macrophages was inhibited by in vitro exposure of the macrophages to acid. CONCLUSIONS: We conclude that the two components of gastric aspiration have diverse effects on local and systemic macrophages. Although there is a synergy between acid and gastric particulate in producing an acute lung injury, the modulatory effects of these injuries on the alveolar macrophages are averse.     

Evaluation of a simplified dual-platform flow cytometric method for measurement of lymphocyte subsets and T-cell maturation phenotypes in the population of Nouna, Burkina Faso

In the context of a larger clinical study in Nouna, Burkina Faso, we evaluated a simplified dual-platform (DP) flow cytometric (FCM) method that allows the determination of major lymphocyte subsets in a single test tube. We compared the phenotyping of lymphocytes with DP FCM and simultaneous measurements with standard single-platform (SP) FCM for samples from 177 individuals. Analysis of the comparative measurements revealed that DP FCM systematically underestimates the proportion of NK cells, overestimates the percentage of CD3(+) CD8(+) lymphocytes, and yields proportions of B cells and CD4(+) T cells comparable with the results from SP FCM. Bland-Altman analysis showed a low bias between both methods and an acceptable precision for percent values of CD4(+) T cells (bias +/- precision, -1% +/- 6%) and CD8(+) T cells (-3% +/- 6%). The absolute cell numbers of all lymphocyte subpopulations, however, were systematically biased towards lower values being obtained by DP FCM. Reference values for the distribution of T-cell maturation phenotypes in 177 healthy adults were calculated using DP FCM. The mean +/- standard deviation (SD) CD4(+)-to-CD8(+) T-cell ratio was 1.61 +/- 0.61, the mean percentage +/- SD of CD4(+) T cells was 42% +/- 7%, and that of CD8(+) T cells 29% +/- 7%. Among CD4(+) lymphocytes, 28% +/- 7% were classified as central memory (CD45RA(low) CCR7(+)), 22% +/- 10% as na?ve (CD45RA(high) CCR7(+)), 45% +/- 12% as effector memory (CD45RA(low) CCR7(-)); and 5% +/- 3% as terminally differentiated effector memory expressing CD45RA (CD45RA(high) CCR7(-)). Among CD8(bright) lymphocytes, 3% +/- 2% had a central memory phenotype, 27% +/- 13% were na?ve, 37% +/- 13% had an effector memory phenotype, and 34% +/- 12% were terminally differentiated effector memory cells expressing CD45RA.     

Bromodeoxyuridine incorporation and Ki 67 expression in oral hairy leukoplakia

OBJECTIVES: Oral hairy leukoplakia (HL) is an acan-thotic, hyperparakeratotic lesion characterised by the presence of a replicative Epstein-Barr virus (EBV) infection in the superficial and adjoining layers of the epithelium. EBV or its gene products are capable of modifying epithelial differentiation. The aim of this study was to establish whether the presence of EBV was associated with an alteration in cell turnover by assessing bromodeoxyuridine (BrdU) incorporation and Ki 67 expression in lesional tissue and control mucosa.
METHODS: Biopsies of HL together with age, site and sex matched controls ( n = 7 and 8 respectively) were incubated in 200 μM BrdU in vitro , fixed in methacarn and processed to paraffin wax. Following acid hydrolysis, incorporated BrdU and Ki 67 were identified in serial 5 fim sections using a three-stage immunoperoxidase technique and cell density expressed as the number of positive cells per mm basement membrane length.
RESULTS: Overall, there was no difference in the number of BrdU positive cells per mm basement membrane length between control and HL tissue. However, within HL alone, the presence'of focal EBV replication was associated with a significant reduction in the number of basal cells incorporating BrdU compared to adjacent EBV free areas (P < 0.05). There was no significant difference between Ki 67 positive cells in control and HL tissue and no evidence of a reduction of Ki 67 positive cells in areas associated with EBV replication.
CONCLUSIONS: These results suggest that there is no evidence of a generalised alteration of the proliferative capacity of basal cells in HL, although the focal reduction in BrdU incorporation may reflect subtle changes on cell turnover by EBV infection.     

FM sonography in diffuse liver disease: prospective assessment and blinded analysis

FM sonography - a signal-processing technique that uses frequency and phase information as well as amplitude data - shows promise in evaluation of patients with diffuse liver disease. In a prospective blinded review of 37 patients with biopsy-proved liver disease and 42 healthy volunteers, FM sonography was clearly superior to traditional amplitude-based (AM) sonography in distinguishing healthy from diseased subjects. Statistically significant differences were seen in accuracy (FM, 98.7%; AM, 84.8%), sensitivity (FM, 97.3%; AM, 70.3%), and negative predictive value (FM, 97.7%; AM, 78.8%). Our data also suggest that current FM sonographic techniques cannot differentiate among histologic findings associated with different hepatic parenchymal abnormalities. It is unclear, therefore, whether FM imaging can reduce the numbers of patients who require biopsy for diagnosis or the frequency of biopsy procedures in patients with known disease.