The effectors of killer activity induced by interferon-alpha and gamma in peripheral blood mononuclear cells

The nature of effectors of interferon (IFN)-alpha or IFN-gamma-induced killer cell activity remains unclear. The aim of this study was to examine killer cell activity induced by IFN-alpha alone, IFN-gamma alone or a combination of both in patients with renal cell carcinoma (RCC) and to determine the phenotypic patterns of these effectors. The study group included 14 patients (12 men and 2 women, median age 64 years, range 36-77) with confirmed RCC. Peripheral blood mononuclear cells (PBMC) from RCC patients or normal volunteers were cultured with IFN-alpha alone, IFN-gamma alone or a combination of both. Cytotoxic activity was assayed against ACHN cells. Subpopulations of effector cells in IFN-induced killer cell activity were characterized by cell sorting. The most effective type of IFN and the optimal concentration of IFN necessary to induce the maximal killer cell activity varied among RCC patients. The killer activity induced by a combination of IFN-alpha and IFN-gamma was significantly greater than that induced by IFN-alpha or IFN-gamma alone. The greatly increased killer activity induced by IFN-alpha and IFN-gamma was seen in the subpopulations CD3(-) CD16(+), CD3(-) CD56(+) and subpopulation CD3(+)CD4(-), CD3(-)CD16(+), CD3(-)CD56(+), CD57(+)CD16(-), respectively. An optimal type of IFN and optimal concentration of IFN seem to increase the effective rate of treatment of RCC. In addition, the role of IFN-alpha seems to be different from that of IFN-gamma in host defense against RCC. A combination treatment with IFN-alpha and IFN-gamma seems to be suitable to increase the effective rate if we could reduce the side effects of IFNs.     

Expression patterns of focal adhesion associated proteins in the developing retina.

Adhesive interactions between integrin receptors and the extracellular matrix (ECM) are intimately involved in regulating development of a variety of tissues within the organism. In the present study, we have investigated the relationships between beta(1) integrin receptors and focal adhesion associated proteins during eye development. We used specific antibodies to examine the distribution of beta(1) integrin ECM receptors and the cytoplasmic focal adhesion associated proteins, talin, vinculin, and paxillin in the developing Xenopus retina. Immunoblot analysis confirmed antibody specificity and indicated that beta(1) integrins, talin, vinculin, and paxillin were expressed in developing retina and in the retinal-derived Xenopus XR1 glial cell line. Triple-labeling immunocytochemistry revealed that talin, vinculin, paxillin, and phosphotyrosine proteins colocalized with beta(1) integrins at focal adhesions located at the termini of F-actin filaments in XR1 cells. In the retina, these focal adhesion proteins exhibited developmentally regulated expression patterns during eye morphogenesis. In the embryonic retina, immunoreactivities for focal adhesion proteins were expressed in neuroepithelial cells, and immunoreactivity was especially strong at the interface between the optic vesicle and overlying ectoderm. At later stages, these proteins were expressed throughout all retinal layers with higher levels of expression observed in the plexiform layers, optic fiber layer, and in the region of the inner and outer limiting membrane. Strong immunoreactivities for beta(1) integrin, paxillin, and phosphotyrosine were expressed in the radially oriented Müller glial cells at later stages of development. These results suggest that focal adhesion-associated proteins are involved in integrin-mediated adhesion and signaling and are likely to be essential in regulating retinal morphogenesis.     

Detection of cytomegalovirus in urine samples by an enzyme-linked immunosorbent assay using a monoclonal antibody against the viral 150-kilodalton protein.

McKeating et al. (J.A. McKeating, P.D. Griffiths, and J.E. Grundy, J. Gen. Virol. 68:785-792, 1987; J. A. McKeating, J. E. Grundy, Z. Varghese, and P. D. Griffiths, J. Med. Virol. 18:341-348, 1986; J. A. McKeating, S. Stagno, P. R. Stirk, and P. D. Griffiths, J. Med. Virol. 16:367-373, 1985) reported previously that beta 2 microglobulin inhibits the detection of human cytomegalovirus (CMV) in urine specimens by an enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody against the glycoprotein of CMV. They postulated that beta 2 microglobulin binds to the viral glycoproteins and masks the antigenic determinants. We developed here an ELISA method for the detection of CMV in urine by using a monoclonal antibody against the viral 150-kDa protein to capture the viral antigen. This assay detected CMV both in culture medium and in urine specifically at concentrations higher than 10(3) PFU/ml and quantitatively at concentrations higher than 10(4) PFU/ml. The sensitivity of the ELISA increased about 10-fold when peroxidase-labeled F(ab')2 from goat anti-human immunoglobulin G was used as a secondary detecting antibody in combination with concentration of the virus in urine samples by ultracentrifugation. The inhibition of ELISA by beta 2 microglobulin was not observed in this ELISA system. When 56 urine specimens from renal transplant recipients were examined for CMV antigens, the ELISA system had a sensitivity of 78% and a specificity of 97%. The positive and negative predictive values of the assay were 95 and 86%, respectively. Furthermore, CMV antigens in urine were quantitated by the assay during the course of typical CMV disease of renal transplant recipient. These results suggest strongly that the measurement of CMV antigens in urine by our rapid and quantitative ELISA system provides very useful data for the monitoring of CMV infections in renal transplant recipients and making decisions about therapy.     

In vivo study of the effects of cryopreservation on heart valve xenotransplantation.

BACKGROUND: Recent reports have suggested that cryopreservation reduces the immunogenicity of donor tissue. The immunomodulation by cryopreservation might influence on the tissue durability after xenotransplantation. We investigated the in vivo morphologic changes in cryopreserved xenograft (CXG) heart valves. MATERIAL AND METHOD: We transplanted a fresh (fresh xenograft; FXG) and a cryopreserved (CXG) porcine aortic root and a cryopreserved canine (cryopreserved allograft; CAG) aortic root into the abdominal aorta of a dog without any immunosuppressive agents. Explanted grafts on the 21st to 49th days after implantation were analyzed morphologically with light microscopy using some special stains, immunohistochemical analysis, and scanning electron microscopy (SEM). RESULT: Light microscopy showed the absence of smooth muscle cells in the media of the aorta in any group after transplantation. FXG valves did not maintain any cellularity after transplantation. CXG valves contained cellular infiltration in themselves. CAG valves contained numerous fibroblasts, which showed the maintenance of tissue integrity without allowing cellular infiltration. The structure of elastic fibers was well maintained, even in the part of CXG valve with cellular infiltration. Immunohistochemical studies documented the infiltration of T lymphocytes in CXG valves that were labeled by anti-CD3 antibodies. SEM demonstrated that no endothelia were seen on the surface of the valves in any group after transplantation. CONCLUSION: We concluded that the cryopreservation method might provide an immunomodulation of xenogeneic heart valves for transplantation.     

Association of HLA alleles with response (especially biochemical response) to interferon therapy in Japanese patients with chronic hepatitis C.

The response of chronic hepatitis C to interferon (IFN) treatment is classified as complete response (CR), biochemical response (BR), or no response (NR). Several studies have found no difference in prevention of hepatocellular carcinoma by IFN therapy between patients with CR and those with BR. We investigated whether specific human leukocyte antigen (HLA) alleles were associated with response to IFN, especially BR, in 138 patients with chronic hepatitis C. Comparing patients with and without CR, male, a low viral titer, genotype 2a or 2b, HLA-B55, and HLA-DRB1-0803 were more common in the group with CR. Multivariate analysis showed that age (adjusted odds ratio [OR], 0.95 by every year [95% confidence interval [CI] 0.90 - 0.99], p = 0.028), genotype 2a or 2b (5.21 [95% CI 1.63 - 16.6], p = 0.005), and low viral titer (8.58 (2.66 - 27.7), p < 0.001) were associated with CR. Comparing patients with BR and NR, the pretreatment alanine aminotransferase (ALT) level was lower in the BR group (p < 0.001). Both HLA-B7 and HLA-DRB1-0101 were more common in this group (p = 0.002). As the alleles HLA-B7 and HLA-DRB1-0101 were in linkage disequilibrium, the HLA-B7-DRB1-0101 haplotype may be associated with BR. Multivariate analysis indicated that a low ALT level (0.98 by every 1 IU/L [95% CI 0.98 - 0.99], p = 0.001) and HLA-B7-DRB1-0101 haplotype (32.3 [95% CI 1.50 - 693.1], p = 0.026) contributed significantly to BR. This study suggested that host HLA expression, but not viral factors, can influence BR.     

Mechanomyography of the human quadriceps muscle during incremental cycle ergometry

The mechanical activity of the human quadriceps muscle during maximal incremental cycle ergometry was investigated by mechanomyography (MMG). MMG and surface electromyography (EMG) recordings of vastus lateralis muscle activity were obtained from nine males. Cycle ergometry was performed at 60?rev/min and work load was incremented step wise by 20?W (3.2?Nm) every minute until volitional fatigue. The mean amplitudes of MMG (mMMG) and EMG (mEMG) during the contraction phase were calculated from the last six contractions in each load. The duration, load and work rate of exercise at exhaustion were 13.3 (1.6)?min, 44.1 (5.5)?Nm, 276.7 (34.7)?W, respectively. A linear relationship between mMMG and load was evident in each subject (r?=?0.868–0.995), while mEMG seemed to dissociate as the load became greater. In the grouped mean data, mMMG was linearly related to load whether aligned to the absolute (r?=?0.995) or maximal (r?=?0.995) load. Involvement of the noise component was further investigated by studying passive cycling by four subjects. Pedals were rotated passively for the first half of each stage (PAS) and the subject then pushed the pedals for the second half (ACT). In the lighter load region, the mMMG of ACT was as small as that of PAS. However, the change in the mMMG of PAS was very small compared with that of ACT. In conclusion, this study demonstrates a linear relationship between the mMMG of the quadriceps muscle and work load during maximal incremental cycle ergometry. The effect of movement noise was thought to be small and stable.     

Short-term desensitization of muscarinic K+ channel current in isolated atrial myocytes and possible role of GTP-binding proteins

The short-term desensitization of the acetylcholine (ACh)-induced K⁺ channel current was examined in single atrial cells of guinea-pig heart. The tight-seal whole cell voltage clamp technique was used. The solution in the pipettes contained GTP or guanosine-5-O-(3-thiotriphosphate) (GTP-S, a non-hydrolyzable GTP analogue). In GTP-loaded cells, ACh evoked a specific K⁺ channel current via GTP-binding proteins (G) in a dose-dependent manner. The K⁺ current showed agonist-dependent desensitization similar to those reported in other cardiac tissues (Nilius 1983; Carmeliet and Mubagwa 1986). The cellular response to ACh was also desensitized by activation of P1-purinergic receptors with adenosine (Ado). In GTP-S-loaded cells, the K⁺ current was gradually induced even in the absence of agonists, probably due to direct activation of G proteins by GTP-S. In the early phase of the spontaneous current increase, ACh evoked a large current transiently. As the GTP-S-induced activation of the current progressed, the magnitude of the ACh-evoked current transient became smaller and finally negligible. Similar results were obtained when Ado was used as an agonist instead of ACh to induce the K⁺ current. Therefore, it is indicated that the agonistreceptor interaction may not be essential for the desensitization of ACh-induced K⁺ current in atrial myocytes.     

Studies on in vitro evaluation for the biocompatibility of various biomaterials: inhibitory activity of various kinds of polymer microspheres on metabolic cooperation

Gap junctional intercellular communication is a function that plays an important role in maintaining cell and tissue homeostasis and in regulating cell growth, development, and differentiation. Change in this function when contacting fibroblasts with various polymer microspheres was estimated using the metabolic cooperation assay system. When the cells were in contact with the microspheres after their adhesion onto a substrate, the function did not alter. However, when they were in contact with precoated microspheres on test dishes, the function was inhibited as the quantity of microspheres increased. Moreover, the inhibition level increased as the diameters of polyethylene and polystyrene microspheres decreased. However, no inhibition was observed if precoated microspheres were composed from poly(L-lactic acid). These findings suggest that the size and the material of microspheres, and how cells recognize the microspheres, are factors affecting cell function of gap junctional intercellular communication. Therefore, estimating this function may provide valuable information about the biocompatibility of many kinds of materials even in the form of particles.     

Distribution of Antigens Detected with MB1, MB2 and MB3 on Non-hematopoietic Human Organs and Various Tumors

We have examined the distribution of antigens detected by MB1, MB2 and MB3 on non-hematopoietic normal human tissues and various types of benign and malignant tumors. MB1 and MB2 reacted with various organs, such as the epithelium of various glands, smooth muscle cells, vascular endothelial cells, and peripheral nerve tissue. The distributions of these two antibodies were essentially identical. Reactivity with MB3 was confined to the ductal eDithelium of salivary glands, the pancreas, and sweat glands, and the cortex of the adrenal gland. lmmunoblotting analysis demonstrated that MB1 and MB2 reacted with a few bands of an extract of myometrial cytoskeletal fraction and salivary gland cytosol fraction, whereas MB3 failed to show any bands on these materials. The reactivities of MB1 and MB2 with various neoplasms were similar to those in normal organs, with slight variations of staining pattern and preponderance in well differentiated tumors. Exceptionally, carcinoid tumor and small round cell tumors, such as small cell carcinoma or neuroblastoma, were not reactive with MB1 and MB2. MB3 reacted with several cases of well differentiated benign and malignant epithelial tumors in various organs, and exceptional cases of malignant schwannoma and glioma. These results indicate that the antigens detected by MB1 and MB2 are distributed broadly on non-hematopoietic normal organs, whereas those detected by MB3 are confined to exceptional cases of epithelial and non-epithelial tumors. Thus, although the use of MB1, MB2 and MB3 is of little value for differential diagnosis of various tumors, these three antibodies may be useful for determining of the origin of some tumor types. Acta Pathol Jpn 42: 339–346, 1992.     

High incidence of pre-excitation syndrome in Japanese families with Leber's hereditary optic neuropathy

Cardiac conduction abnormalities have been reported in families with Leber's hereditary optic neuropathy (LHON). The pre-excitation syndrome, Wolff-Parkinson-White syndrome or Lown-Ganong-Levine syndrome, is reportedly common in Finns with LHON, being seen in 14 (9%) of the 163 individuals with mitochondrial DNA (mtDNA) mutations. While this syndrome is thought to be rare in other ethnic groups with LHON, the present study of 35 Japanese LHON families confirmed that it is also relatively common among Japanese families, being seen in 5 (8%) of the 63 individuals with mtDNA mutations. It remains to be determined whether the high incidence of the pre-excitation syndrome in Finnish and Japanese LHON families is due to a particular genetic composition of ethnic groups such as in Finland and in Japan, or only to a reporting bias.