Histamine N-methyltransferase inhibitor potentiates histamine- and antigen-induced airway microvascular leakage in guinea pigs

Background: Histamine N-methyltransferase (HMT) modulates histamine- and antigen-induced bronchoconstriction. However, it is unclear whether vascular permeability evoked by an allergic reaction can be exaggerated by inhibition of HMT activity. Methods: We studied the effects of intravenously injected SKF 91488, a specific HMT inhibitor, on increases in plasma extravasation induced by intravenously injected histamine in unsensitized guinea pigs and by intravenously injected ovalbumin antigen in guinea pigs sensitized to ovalbumin in vivo with Evans blue dye as a marker. Results: Pretreatment with SKF 91488 shifted, in a dose-dependent fashion, the dose-response curves of the leakage of dye to histamine to lower concentrations in the trachea, main bronchi, and nasal mucosa. Likewise, pretreatment with SKF 91488 (20 mg/kg intravenously) significantly increased the leakage of dye induced by ovalbumin antigen (200 μg/kg intravenously) in three parts of the airway (p < 0.05). In contrast to SKF 91488, intravenously injected aminoguanidine, a specific inhibitor of diamine oxidase (16 mg/kg intravenously), did not alter the leakage of dye induced by histamine (from 0.001 μg/kg to 10 μg/kg intravenously) (p > 0.20). HMT activities were observed in the nasal mucosa, as well as in the trachea and main bronchi, as shown in a previous study. Conclusion: These findings suggest that HMT modulates the effects of exogenous histamine and endogenously released histamine induced by antigen challenge on plasma extravasation in the airway in guinea pigs in vivo. (J ALLERGY CLIN IMMUNOL 1995;96:910-6.)     

Neutralizing activity of the antibodies against two kinds of envelope glycoproteins of Sendai virus

Summary Murine monoclonal antibodies against the fusion (F) and hemagglutininneuraminidase (HN) proteins of Sendai virus (SV) were prepared and studied on their antiviral activities, particularly on the neutralization of infectivity. On the analysis with solid phase competitive ELISA, 26 anti-HN antibodies were divided into at least four groups (HN-I, -II, -III and -IV). Antigenic sites recognized by the HN-I, -II, and -III group antibodies topographically separate from each other. Sites recognized by the HN-IV group antibodies overlaps partially with ones recognized by the HN-I, HN-II and -III group antibodies. The antibodies belonging to the HN-III group highly neutralize the infectivity of SV and weakly or not at all inhibit the hemagglutination (HA). In contrast, the HN-IV group antibodies strongly inhibit HA, but weakly neutralize the infectivity. Adsorption of SV to chicken red blood cells or L cells is inhibited by the HN-IV antibodies, but scarcely by the HN-III antibodies. On the other hand, incubation with HN-III antibodies of HeLa cells that have been preadsorbed with SV at 4° C, followed by culture at 37° C, causes inhibition of infection, but the HN-IV antibodies do not effectively interfere with such infection.The competitive ELISA showed that 17 anti-F antibodies were divided into two groups (F-I and -II). Two antigenic sites recognized by the antibodies, however, seem to be near to each other because a certain competition is observed between the antibodies of both groups. Two of the seven antibodies belonging to the F-II group inhibit the hemolysis activity and also neutralize the infectivity of SV, but the other five F-II antibodies do not. One of the anti-F antibodies has a low HI activity, and, in competition tests, competes with one of the anti-HN antibodies (HN-IV).With 2 Figures     

Kinetic profiles of sequential gene expressions for chemokines in mice with contact hypersensitivity

Using cDNA microarray technology, the expression of chemokine genes in the elicitation site of 2,4,6-trinitrochlorobenzene-induced contact hypersensitivity (CHS) was examined in mice. Of the 33 genes analyzed, levels of 11 gene expressions changed, and these can be assigned to four groups based on their kinetic patterns; (1) LARC/CCL20 whose mRNA level increased rapidly at 3 h post-challenge and then gradually decreased, (2) JE/CCL2, MARC/CCL7, MIP-1gamma/CCL9, monocyte chemoattractant protein (MCP)-5/CCL12, ELC/CCL19 and BRAK/CXCL14 whose mRNA levels increased with time and reached the maximum at 6-9 h post-challenge, (3) LIX/CXCL5, Mig/CXCL9 and IP-10/CXCL10 whose mRNA levels increased gradually at least up to 12 h post challenge, and (4) SLC/CCL21 whose mRNA level decreased gradually with time after challenge. The findings suggest that sequential expression of chemokine genes is essential for orientating non-specific skin response to hapten-specific CHS response through the recruitment of inflammatory cells such as neutrophils, monocytes/macrophages and T-cells from the circulation into the tissue site.     

Detection of human papillomavirus DNA in invasive cervical cancers by the polymerase chain reaction and its clinical significance.

In order to detect human papillomavirus (HPV) DNA in invasive cervical cancers, three different polymerase chain reactions to amplify different subgenomic fragments of HPV DNA were carried out on DNA extracted from 93 formalin-fixed and paraffin-embedded tumor tissues. This study detected HPV DNA in 54 cases (58.1%), which broke down to HPV 16 in 39 (41.9%) cases, HPV 18 in six (6.4%), HPV 52 in three, HPV 33 in one and unclassified HPV type in the remainder. Histopathologically, squamous cell carcinomas frequently contained HPV 16, whereas, HPV 18 was present in adenocarcinoma, adenosquamous cell carcinoma and small cell carcinoma of the cervix. Clinicopathological study revealed that HPV 16 and 18 DNA found were more frequently than other HPV subtypes in premenopausal patients. Moreover, HPV 18 DNA-positive cancers had a relatively high recurrence rate. These results indicate that cervical cancers might be clinically influenced by the difference in subtypes of the infecting HPV.     

A case of malignant duct-islet cell tumor of the pancreas immunohistochemical and cytofluorometric study.

A rare duct-islet cell tumor of the pancreas was studied using immunohistochemical, cytofluorometric and histochemical methods. Histology and immunohistochemistry revealed that the tumor contained two distinct cell types; islet cell-like neuroendocrine cells and exocrine duct cell components, suggesting an endodermal origin for both types. The cells showed marked pleomorphism an vascular and perineural invasion at the tumor periphery. Cytofluorometric study of the tumor cell DNA revealed an increased mean nuclear DNA content, without any aneuploidy. Histochemically, the tumor cells contained an increased number of argyrophilic nucleolar organizer regions (AgNORs) in their nuclei. The malignant potential of this duct-islet cell tumor was suggested.     

Expression and characterization of mouse angiotensin II type 1a receptor tagging hemagglutinin epitope in cultured cells

The octapeptide angiotensin II mediates the physiological actions of the renin-angiotensin system through activation of several angiotensin II receptor (AT) subtypes, in particular AT1 (AT1a and AT1b in the case of rodents). Although we and others have generated mutant mice in which the AT1a gene was disrupted, the function of mouse AT1 remains to be fully elucidated, due to the lack of effective tools involving antibodies against AT1 for detecting biological responses in cellular conditions. To avoid these problems, we constructed the hemagglutinin (HA)-tagged mouse AT1a, and stably introduced this recombinant receptor into human embryonic kidney 293-T cells. Radioligand binding of [(125)I] angiotensin II to AT1a was specific, saturable, and reversible. Scatchard analysis demonstrated that the transfected receptor had a dissociation constant of 1.7 nM with a density of 1.2 x 10(5) sites/cells. Angiotensin II stimulated a rapid increase in cytosolic free calcium, and angiotensin II-induced phosphorylation of extracellular signal-regulated kinases (Erk) was found in a dose-dependent manner. After solubilization, Western blot analysis showed specific interactions between an anti-HA antibody and HA-tagged mouse AT1a. Furthermore, a significant proportion of HA-tagged mouse AT1a was specifically immunoprecipitated with this antibody. In the immunocytochemical and electronmicroscopic studies, treatment of this cell line with angiotensin II resulted in decrease in signals of the surface receptors. Based on these results, the cell line established here provides an excellent tool for studying angiotensin II actions mediated through mouse AT1a, at sub-nanomolar concentrations.     

Secreted Reelin molecules form homodimers

During mammalian brain development, neurons are generated along the ventricle, migrate radially, and become aligned in defined patterns. These precise patterns of neuronal alignment are regulated by an extracellular matrix protein Reelin, and binding of Reelin to its receptors induces tyrosine phosphorylation of the intracellular adaptor protein disabled 1 (Dab1). We recently reported that Reelin molecules assemble to form a homomeric protein complex. Although the number of molecules in the full-length complex is unknown, recombinant N-terminal fragments, which contain the epitope for the function-blocking CR-50 antibody, assembled to form a complex of more than 40 monomers. When the N-terminus was deleted from Reelin, the truncated protein did not form a stable complex. To further characterize the Reelin assembly, we performed biochemical analysis of the full-length Reelin assembly in this study. Here, we report that a full-length Reelin forms a disulfide-linked homodimer. A chemical crosslinking experiment on secreted Reelin confirmed that only dimers are formed by the full-length protein. However, interestingly, chemical crosslinking of the N-terminus-truncated Reelin resulted in the formation of larger complexes, in addition to dimers, suggesting that the tertiary structure required for the proper and stable assembly/dimerization was altered by the truncation. The truncated protein did not induce efficient tyrosine phosphorylation of Dab1, although it bound well to the receptors. These findings demonstrate the functional importance of the N-terminal region of Reelin for proper dimerization and signaling. Proper but not simple extracellular crosslinking of the receptors by these dimers may be important for Reelin signaling to occur.     

Disorder in reciprocal innervation upon initiation of voluntary movement in patients with Parkinson's disease

Summary Reciprocal innervation of the soleus motoneurones upon initiation of voluntary ankle dorsiflexion was investigated in eight patients with Parkinson's disease. H-reflex and visually guided step tracking methods were used for testing moto-neurone excitability and for controlling the timing of movement initiation, respectively. While reciprocal inhibition appeared almost simultaneously with the agonist electromyographic (EMG) onset in normal subjects (Kagamihara and Tanaka 1985), facilitation appeared in the majority of patients under the same onset condition. It increased slowly, reaching a maximum at about 100 ms after the EMG onset. It then subsided slowly at around 200–300 ms, and was replaced thereafter by an inhibitory effect. No coactivation of the soleus muscle was detected electromyographically. The facilitation between the EMG onset and the onset of mechanical contraction was attributed to the direct effect of the descending command from the brain, suggesting a certain disorder in controlling the system for reciprocal innervation.     

Transgenic rabbits with increased VEGF expression develop hemangiomas in the liver: a new model for Kasabach-Merritt syndrome

Clinical studies have provided ample evidence that high (either systemic or local) levels of vascular endothelial growth factor (VEGF) are associated with several pathophysiological disorders, including hemangiomas. To investigate whether elevated VEGF expression could directly affect these disorders, we created a transgenic (Tg) rabbit model with increased hepatic expression of the human VEGF(165) transgene under the control of the human alpha-antitrypsin promoter. Tg rabbits exhibited marked hepatomegaly, with livers 2.5-fold heavier than those of control rabbits. Histological analysis revealed that the livers of Tg rabbits showed prominent dilation of the sinusoids and formed various-sized blood vessel networks, a feature of diffuse hemangiomas. Immunohistochemical staining revealed that the hepatocytes produced VEGF(165), whereas plasma VEGF(165) was not detected. Furthermore, Tg rabbits suffered from hemolytic anemia, thrombocytopenia and splenomegaly, which was associated with marked extramedullary hematopoiesis. The manifestations of Tg rabbits mimic many of the features of hemangiomatous disorders in humans such as the Kasabach-Merritt syndrome, and therefore this model may be potentially useful for the study of the pathogenesis and complications of hemangiomas as well as the investigation of angiogenesis inhibitors.