Colocalization Research Articles

Anti-interleukin 8 autoantibody: interleukin 8 immune complexes visualized by laser confocal microscopy in injured lung: Colocalization with FcγRIIa in lung tissue from acute respiratory distress syndrome patients

•Context.-Anti-interleukin 8 autoantibody:interleukin 8 (anti-lL-8 autoantibody:lL-8) complexes are present in lung fluids of patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS), and levels of these complexes correlate with progression to and the outcome of ARDS. FcyRlla, an immunoglobulin G (IgG) receptor, mediates proinflammatory activity of the complexes. Objective.-To evaluate lung tissues from patients with ARDS for presence of anti-lL-8 autoantibody:lL-8 complexes and to establish whether the complexes associate with FcyRlla. Design.-Lung tissue sections from 3 patients with ARDS and sections of normal lung tissues from 3 patients were stained with antibodies against IL-8 and IgG to detect immune complexes and with antibody against FcγRlla. In some experiments, sections were blocked with anti-Fcγ-Rlla antibody before staining. Samples were analyzed using confocal microscopy. Results.-Interleukin 8 costained with IgG and FcγRlla in lung tissues from patients with ARDS but not in control tissues, suggesting that anti-lL-8 autoantibody:lL-8 complexes are deposited in lungs of patients with ARDS via FcγRlla. Further, colocalization between IL-8 and FcγRlla could be blocked by anti-FcγRlla. Conclusions.-Our data demonstrate that anti-lL-8 autoantibody:lL-8 complexes are present in lung tissues of patients with ARDS, and are attached to FcγRlla.

Expression and localization of endothelin-converting enzyme-1 isoforms in human endothelial cells.

Endothelin-converting enzyme (ECE)-1 is a membrane-bound metalloprotease responsible for production of vasoactive endothelin (ET)-1 from inactive big ET-1. ECE-1 exists as four separate isoforms, ECE-1a, b, c, and d, which differ only in their amino-terminal regions. We investigated the expression and localization of the ECE-1 isoforms in primary human umbilical vein endothelial cells (HUVECs) and EAhy926 cells. Reverse transcriptase polymerase chain reaction showed expression of all four isoforms in both cell lines, with ECE-1d seeming, at least qualitatively, to be the predominant isoenzyme. Isoform-specific polyclonal antibodies were used to investigate isoform protein expression. ECE-1a, b, and c protein was detected in EAhy926 cells by immunoblotting; only ECE-1a and ECE-1c were detected in HUVECs. Using immunofluorescence microscopy analysis, both HUVEC and EAhy926 cells showed nuclear immunoreactivity with a monoclonal antibody recognizing all ECE-1 isoforms. The ECE-1a antibody also showed nuclear immunoreactivity in both cell lines; this seemed to colocalize with nucleolin. The ECE-1b antibody showed nuclear immunoreactivity in EAhy926 cells, but no overlap with nucleolin was seen. Intracellular immunoreactivity was seen in both cell lines using the ECE-1c antibody; this showed some colocalization with concanavalin A (an endoplasmic reticulum marker). von Willebrand Factor was used as a marker for Weibel-Palade bodies in HUVECs, but no colocalization with ECE-1 was seen during this study. The data presented here sheds new light on the localization of ECE-1a, b, and c in cultured human endothelial cells, which may further understanding of the ET system and aid design of therapeutic ECE inhibitors.

Combined In Situ Hybridization and Immunohistochemistry for Automated Detection of Cytomegalovirus and p53.

Background: Cytomegalovirus (CMV) infection has been shown to be associated with p53 overexpression in coronary artery restenosis. We investigated the occurance of this association in other forms of CMV infection using an automated in situ hybridization (ISH) technique. Methods and Results: We performed ISH for CMV using digoxigenin-labeled or biotinylated probes followed by avidin-alkaline phosphatase and nitroblue tetrazolium color substrate. Immunohistochemistry (IHC) was then performed using an anti-p53 antibody utilizing streptavidin-immunoperoxidase and 3,3;-diaminobenzidine tetrahydrochloride as a chromogen. Sixteen cases with characteristic cytomegalic inclusions from a variety of body sites were examined. All 16 cases were positive for CMV by ISH. Nine of sixteen expressed nuclear p53. Six of these nine cases showed viral cytopathic effect in the cells with p53 expression. In an illustrative case, double colocalized staining for CMV and p53 protein was demonstrated in individual cytopathic cells. When microwave antigen retrieval was necessary, ISH was performed before IHC, and our standard microwaving time was reduced by two-thirds. Conclusions: The colocalization of p53 protein overexpression with CMV within single cells adds further evidence that this overexpression is a viral-induced phenomenon. The combined ISH and IHC assay can be carried out in a rapid automated mode, increasing the ease of investigating relationships between message and protein expression within single cells in a wide variety of settings.

Recurrent chromosomal rearrangements at bands 8q24 and 11q13 in gastric cancer as detected by multicolor spectral karyotyping.

To identify chromosomal translocations specific to gastric cancer (GC), spectral karyotyping (SKY) analysis was performed on established cell lines and cancerous ascitic fluids. SKY analysis of 10 established cell lines and seven cancerous ascitic fluid samples identified recurrent chromosomal breakpoints and translocations in GC, several of which involved chromosomal loci of oncogenes or tumor suppressor genes. A total of 630 chromosomal breaks were identified. Chromosome no.8 was the most frequently involved in rearrangements (65 breaks), followed by chromosomes no.11 (53), no. 1 (49), no. 7 (46), no. 13 (37), no. 3 (36), no. 17 (33), and no. 20 (29). Frequent breakpoints were detected in 8q24.1 (30 breaks), 11q13 (29), 13q14 (16), 20q11.2 (14), 7q32 (13), 17q11.2 (13), 18q21 (12), 17q23 (9), 18q11.2 (9). SKY analysis identified a total of 242 chromosomal rearrangements including 190 reciprocal and non-reciprocal translocations. The recurrent combinations of chromosomal bands involved in translocations were 8q24.1 and 13q14 (3 cases), 8q24.1 and 11q13 (3), 11q13 and 17q11.2 (2), and 18q11.2 and 20q11.2 (2). Our study validated the ability of SKY to characterize in detail the chromosomal rearrangements in solid tumors and derived cell lines. Moreover, fluorescence in situ hybridization helped to identify the insertions, translocations, and homogeneously staining regions of MYC and CCND1 gene loci. The non-random co-localization of certain cytogenetic bands suggests the importance of chromosomal translocations in gastric carcinogenesis, by serving as landmarks for the cloning of GC causing genes.

Xenon induced preconditioning involves increased HSP27 translocation and actin-HSP27 colocalisation

Xenon induced preconditioning involves increased HSP27 translocation and actin-HSP27 colocalisation

Quantitative genomics: exploring the genetic architecture of complex trait predisposition.

Most phenotypes with agricultural or biomedical relevance are multifactorial traits controlled by complex contributions of genetics and environment. Genetic predisposition results from combinations of relatively small effects due to variations within a large number of genes, known as QTL. Well over 200 QTL have been reported for growth and body composition traits in the mouse, which likely represent at least 50 to 100 distinct genes. Molecular biology has yielded significant advances in understanding these traits at the metabolic and physiological levels; however, little has been learned regarding the identity and nature of the underlying polygenes. In addition to the significantly poor precision inherent to QTL localization, it is very difficult to differentiate between co-localization and coincidence when comparing QTL with other QTL and with potential candidate genes. The wide gap between our knowledge of physiological mechanisms underlying complex traits and the nature of genetic predisposition significantly impairs discovery of genes underlying QTL. Identification and genetic mapping of key transcriptional, proteomic, metabolomic, and endocrine events will uncover large lists of significant positional candidate genes for growth and body composition. However, integration of experimental approaches to jointly evaluate predisposition and physiology will increase success of QTL identification by merging the power of recombination with functional analysis. Measuring physiologically relevant subphenotypes within a structured QTL mapping population will not only facilitate pathway-specific prioritization among candidate genes, but may also directly identify genes underlying QTL. This would advance our understanding of the genetic architecture of complex traits by testing the central hypothesis that genes controlling predisposition to a quantitative trait are primarily involved in trans-regulation of the primary physiological pathways that regulate the trait.

Involvement of NP95 in G2/M Arrest and Its Colocalization with H2AX afterX-irradiation

Involvement of NP95 in G2/M Arrest and Its Colocalization with H2AX afterX-irradiation

Aberrant Localization of the Neuronal Class III b-Tubulin in Astrocytomas A Marker for Anaplastic Potential

c Background.—The class III b-tubulin isotype (bIII) is widely regarded as a neuronal marker in development and neoplasia. In previous work, we have shown that the expression of bIII in neuronal/neuroblastic tumors is differentiation dependent. In contrast, the aberrant localization of this isotype in certain nonneuronal neoplasms, such as epithelial neuroendocrine lung tumors, is associated with anaplastic potential. Objective.—To test the generality of this observation, we investigated the immunoreactivity profile of bIII in astrocytomas. Design.—Sixty archival, surgically excised astrocytomas (8 pilocytic astrocytomas, WHO grade 1; 18 diffuse fibrillary astrocytomas, WHO grade 2; 4 anaplastic astrocytomas, WHO grade 3; and 30 glioblastomas, WHO grade 4), were studied by immunohistochemistry using anti-bIII monoclonal (TuJ1) and polyclonal antibodies. A monoclonal antibody to Ki-67 nuclear antigen (NC-MM1) was used as a marker for cell proliferation. Antibodies to glial fibrillary acidic protein (GFAP) and BM89 synaptic vesicle antigen/synaptophysin were used as glial and neuronal markers, respectively. Results.—The bIII immunoreactivity was significantly greater in high-grade astrocytomas (anaplastic astrocytomas and glioblastomas; median labeling index [MLI], 35%; interquartile range [IQR], 20%‐47%) as compared with diffuse fibrillary astrocytomas (MLI, 4%; IQR, 0.2%‐21%) (P , .0001) and was rarely detectable in pilocytic astrocytomas (MLI, 0%; IQR, 0%‐0.5%) (P , .0001 vs high-grade astrocytomas; P , .01 vs diffuse fibrillary astrocytomas). A highly significant, grade-dependent relationship was observed between bIII and Ki-67 labeling and malignancy, but this association was stronger for Ki-67 than for bIII (bIII, P , .006; Ki-67, P , .0001). There was co-localization of bIII and GFAP in neoplastic astrocytes, but no BM89 synaptic vesicle antigen/synaptophysin staining was detected. Conclusions.—In the context of astrocytic gliomas, bIII immunoreactivity is associated with an ascending gradient of malignancy and thus may be a useful ancillary diagnostic marker. However, the significance of bIII-positive phenotypes in diffuse fibrillary astrocytomas with respect to prognostic and predictive value requires further evaluation. Under certain neoplastic conditions, bIII expression is not neuron specific, calling for a cautious interpretation of bIII-positive phenotypes in brain tumors. (Arch Pathol Lab Med. 2001;125:613‐624)

Immunolocalization of NBC3 and NHE3 in the rat epididymis: colocalization of NBC3 and the vacuolar H+-ATPase.

In the male reproductive tract, the epididymis plays an important role in mediating transepithelial bicarbonate transport and luminal acidification. In the proximal vas deferens, a significant component of luminal acidification is Na+-independent, and mediated by specific cells that possess apical vacuolar proton pumps. In contrast, luminal acidification in the cauda epididymidis is an Na+-dependent process. The specific apical Na+-dependent H+/base transport process(es) responsible for luminal acidification have not been identified. A potential clue as to the identity of these apical Na+-dependent H+/base transporter(s) is provided by similarities between the transport properties of the epididymis and the mammalian nephron. Specifically, the H+/base transport properties of caput epididymidis resemble the mammalian renal proximal tubule, whereas the distal epididymis and vas deferens have characteristics in common with renal collecting duct intercalated cells. Given the known expression of the Na+/H+ antiporter, NHE3, in the proximal tubule, and of the electroneutral sodium bicarbonate cotransporter, NBC3, in renal intercalated cells, we determined the localization of NHE3 and NBC3 in various regions of rat epididymis. NBC3 was highly expressed on the apical membrane of apical (narrow) cells in caput epididymidis, and light (clear) cells in corpus and cauda epididymidis. The number of cells expressing apical NBC3 was highest in cauda epididymidis. The localization of NBC3 in the epididymis was identical to the vacuolar H+-ATPase. The results indicate that colocalization of NBC3 and the vacuolar H+-ATPase is not restricted to kidney intercalated cells. Moreover, the close association of the two transporters appears to be a more generalized phenomenon in cells that express high levels of vacuolar H+-ATPase. Unlike NBC3, NHE3 was most highly expressed on the apical membrane of all epithelial cells in caput epididymidis, with less expression in the corpus, and no expression in the cauda. These results suggest that apical NBC3 and NHE3 potentially play an important role in mediating luminal H+/base transport in epididymis.

MRI predicts area of increased plasminogen activation in permanent focal cerebral ischemia

P117 Background: Increased proteolytic activity after ischemic stroke may promote brain edema and secondary hemorrhage. Increased plasminogen activation has previously been detected in infarcted regions after focal cerebral ischemia. In our study, we investigated whether areas of increased plasminogen activation can be related to and therefore later be predicted by intravital magnetic resonance imaging. Methods: Permanent occlusion of the middle cerebral artery (MCA) in male Fisher rats was achieved by electrocoagulation after subtemporal craniectomy. After 48 hours the extent of the ischemic lesion was determined by T 2 -weighted in vivo MRI. The animals were sacrificed after 72 (n=4) and 168 (n=4) hours. Areas of plasminogen activation were then detected in 10 μm brain slices by in situ zymography using an overlay containing plasminogen and the plasmin substrate casein. The area of the MRI lesion was compared to the area of increased plasminogen activation in respective brain sections using an advanced videoimaging system. Results: MRI at 48 hours consistently revealed extensive lesions affecting most of the basal ganglia and the cortex (84.9 ± 8.6 % and 86.6 ± 10.1 %, respectively). After 72 hours of permanent ischemia, plasminogen activation in respective sections measured 67.5 ± 14.6 % and 80.5 ± 6.8 % of total basal ganglia and cortex area. After 168 hours respective values were 81.8 ± 7.3 % and 88.1 ± 2.7 %. There was no significant difference in the extent of plasminogen activation between the two time intervals. Comparing individual animals, MRI lesion and plasminogen activation showed close overlapping. This colocalisation was significantly higher than expected if areas were randomely distributed (p

Colocalization of somatostatin- and HLA-DR-like immunoreactivity in dendritic cells of psoriatic skin.

This study presents an immunohistochemical characterization of somatostatin-positive dendritic cells in psoriatic lesions. Somatostatin is a neuropeptide with inhibitory action on several neuropeptides and hormones, but also with immunomodulating properties, and has been used in several studies as treatment for psoriasis. The number of somatostatin-positive dendritic cells was found to be larger in psoriatic lesions than in normal skin of psoriasis patients and healthy controls. Colocalization of somatostatin and HLA-DR immunoreactivity was demonstrated in a subgroup of dendritic cells of psoriatic skin, whereas double-labelled cells were not found in uninvolved skin. The somatostatin-positive cells in the epidermis and dermis did not co-express CD1a, CD35, CD45RB, CD45RO, CD68, factor XIIIa or S-100. On the basis of these findings, the somatostatin-positive cells seem to represent a specific population of dermal dendritic cells, distinct from Langerhans' cells and factor XIIIa-positive cells, which are found in elevated amounts in chronic plaque psoriasis.

Morphological studies on Mn-SOD, NOS and calcium binding proteins in the rat hippocampus.

The immunohistochemical localization of manganese superoxide dismutase (Mn-SOD), nitric oxide synthase (NOS) and calcium (Ca) binding proteins; calbindin-D28K (Calb) and parvalbumin (Parv), was investigated in the rat hippocampus by using a double immunostaining method and an enzyme histochemical staining method. These substances showed considerable regional immunoreactivities in the hippocampus. (1) Although Mn-SOD- and NOS-immunoreactive neurons showed a similar distribution, virtually all of the latter neurons had no or weak Mn-SOD immunoreactivity in all subfields. This finding suggests that NO-producing neurons are not directly associated with Mn-SOD scavenger system in the hippocampus. (2) The co-localization of NOS and Parv or Calb was rarely found throught the hippocampus. (3) Neurons which were intensely immunostained for Mn-SOD were frequently found to be Parv-immunoreactive non-pyramidal cells, or Calb-immunoreactive pyramidal and non-pyramidal cells. From the latter two findings, it is speculated that Ca buffering effects of Parv and Calb are required in Mn-SOD neurons, but not in NO-producing neurons. Since Parv- or Calb-containing non-pyramidal cells are known to be GABAergic, most if not all, Mn-SOD-containing non-pyramidal cells may be GABAergic inhibitory neurons in the rat hippocampus. On the other hand, NOS neurons are also known to belong to GABAergic non-pyramidal cells. Therefore, Mn-SOD and NOS neurons constitute two distinct subclasses of non-pyramidal inhibitory neurons in the hippocampus.

Neuropeptides in the human penis: an immunohistochemical study.

In the present study, the distribution of neuropeptides in the human penis is demonstrated by immunohistochemistry (IHC). IHC screening detected a complex network of nerve fibers containing vasoactive intestinal polypeptide (VIP), peptide histidine-methionine (PHM), prepro-VIP (111-122), neuropeptide Y (NPY), C-flanking peptide of NPY (C-PON), calcitonin gene-related peptide, substance P, and galanin immunoreactivities. Special attention was also given to the recently isolated, VIP-related lizard peptide helospectin, which could also be detected in neuronal elements in the penis. Colocalization studies showed the coexistence of VIP, PHM, and partly helospectin, and of NPY with C-PON within nerve fibers in the cavernous and spongious body, the glans penis, and the urethra.

Parvalbumin immunoreactivity in the telencephalic hemispheres of the tench, Tinca tinca.

The distribution of parvalbumin immunoreactivity in the telencephalic hemispheres of the tench (Tinca tinca L.) was studied using a monoclonal antibody and the avidin-biotin immunoperoxidase method. A wide distribution of immunoreactive structures was found in both dorsal and ventral areas of the telencephalic hemispheres. Normally, the parvalbumin-immunostained neurons in the dorsal area were smaller, more weakly stained, and more scattered than those in the ventral one. In the dorsal area, parvalbumin-immunoreactivity was observed in practically all divisions. The highest density of positive somata was found in the lateral nucleus. Density of stained fibres was generally low. Labelling in the ventral area appeared in the ventral and dorsal nuclei, with a high density of both immunoreactive fibres and cell bodies. Neurons were seen close to the ventricle, arranged in two groups of neurons with different sizes and morphologies. The distribution and morphological characteristics of the parvalbuminergic cells were similar to those of previously described GABAergic elements, suggesting a possible colocalization of both substances in the teleost telencephalon, as it has been observed in different telencephalic areas of land vertebrates. On the other hand, significant differences were seen between the distribution of parvalbumin in the telencephalic hemispheres of the tench in comparison with its distribution pattern in supposed homologous structures of the forebrain of amniotes.

Cerebellar localization and colocalization of GABA and calcium binding protein-D28K.

Immunocytochemical studies using antibodies raised against the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) and against the 28 Kd vitamin D dependent calcium binding protein (calbindin) in the cerebellum, are reviewed. The GABA immunoreactive neurones found in the cerebellar cortex were the Purkinje cell (PC), the three classes of intrinsic inhibitory interneurones, stellate, basket and Golgi cells and the cells of Lugaro. Some of the neurons of the cerebellar nuclei were also found to be GABA immunoreactive. A part of these could be identified as extrinsic neurones projecting either back to the cerebellar cortex, or to the inferior olive, both these pathways being topographically highly organized but arising from independent parent neurons. The presumed inhibitory function of these two pathways are discussed. Calbindin immunoreactivity in the cerebellum was confined to the PCs, staining concerned the whole cell including soma, branching dendrites, axons and axons terminals. The antibody, which appears to be tightly bound to the PC in vivo, failed to stain some of the PC when cerebellar slices maintained in vitro were studied. The stability of the antigen-antibody binding and the use of calbindin as a marker specific for the PC in the cerebellum, is discussed. Co-localization of GABA with calbindin as well as with other calcium binding proteins are reported to be found in the PCs. While these co-localizations have led to much speculation, conclusive functional roles for them have not been identified at present.

Remarks on Colocalization and Equivalence

Remarks on Colocalization and Equivalence

Duality between colocalization and localization

Duality between colocalization and localization

Colocalization and localization

Colocalization and localization