Molecular cloning of agonistic and antagonistic monoclonal antibodies against human 4-1BB.

Previously, we prepared two different monoclonal antibodies (mAbs) against human 4-1BB (CD137): an agonistic mAb BBK-1 and an antagonistic mAb BBK-2. In this paper, we describe the molecular cloning of these two mAbs and present comparisons of their amino acid sequences. cDNAs encoding the heavy (H) and light (L) chains of the two mAbs were cloned by screening of cDNA libraries constructed from hybridomas secreting these mAbs. Comparisons of amino acid sequences of the two mAbs showed that, while the constant regions of the H and L chains were identical between the two mAbs, the variable region showed 45% identity in H chains and 48% identity in L chains. This suggests that these two mAbs recognize different epitopes of 4-1BB and may have different effects on the activity of 4-1BB.     

The contribution of reoxygenation to ischemic brain damage

This study examined the hypothesis that the level of postischemic reperfusion affects the severity of the resulting neuronal necrosis. In rats, tissue PO2% was monitored as an index of flow (reoxygenation) at four cortical sites by chronically implanted platinum electrodes. Twenty minutes of total global cerebral ischemia was followed by 30 min of reoxygenation. The level of reoxygenation was controlled to maintain the PO2 nearly constant at one or more of the cortical electrodes. Tissue from within 400 microns of each of 19 electrode sites among seven rats was evaluated histologically. There was a positive correlation between reoxygenation level and severity of neuronal damage. Perineuronal lucent halo formation, probably representing astrocyte foot process swelling, was negatively correlated with reoxygenation level. This study demonstrates that ischemic neuronal damage was aggravated by increased reoxygenation but that perineuronal swelling, as evidenced by halo formation, was somewhat ameliorated.     

Cytochrome P-45011 beta in rat brain

The presence of cytochrome P-45011 beta in rat brain was studied by immunohistochemistry using polyclonal rabbit antibodies raised against purified bovine adrenocortical P-45011 beta, which is involved in the steroid 11 beta-hydroxylation and glucocorticoid formation. The results showed that cytochrome P-45011 beta immunoreactivity is selectively localized to the tracts of myelinated fibers throughout the brain. The specificity of immunohistochemical stainings with P-45011 beta antibodies was established by control tests including nonimmune rabbit immunoglobulin Gs and P-45011 beta antibodies absorbed with purified antigen. Western immunoblots of homogenates from different brain areas with P-45011 beta antibodies, together with biochemical enzymatic assays for cytochrome P-45011 beta monooxygenase activity in these homogenates, confirmed the selective localization of this enzyme observed with immunohistochemistry. Cytochrome P-45011 beta and 11 beta-hydroxylase activity were detected in a homogenate from the cortical white matter (brain area rich in myelinated fibers) as in that from the rat adrenal, but were not detectable in a homogenate from the cerebral cortex (brain area poor in myelinated fibers). Furthermore, quantitation of the P-45011 beta bands on the immunoblots by the areal density revealed that the cortical white matter contains approximately 1.4 pmol of cytochrome P-45011 beta/mg of tissue protein, the value of which was about one sixth of the corresponding value estimated in the rat adrenal. This relatively high content of cytochrome P-45011 beta was also reflected in a relatively high level of 11 beta-hydroxylase activity measured in a homogenate of this brain area by biochemical enzymatic assays using [4-14C]-11-deoxycorticosterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

The innervation of the levator veli palatini muscle by the glossopharyngeal nerve

We developed a novel method which enables bloodless exposure of the levator veli palatini muscle in rat in order to investigate the physiological properties of this muscle. The levator veli palatini muscle which is innervated by a branch of the glossopharyngeal nerve showed rhythmic spontaneous movement in rats. Cutting the branch supplying LVP of the glossopharyngeal nerve caused cessation of the spontaneous movement of the levator veli palatini muscle. The spontaneous discharges of the glossopharyngeal nerve were synchronized with those of the phrenic nerve. A mixture of 95% oxygen and 5% room air influenced the efferent discharges from the branch of the glossopharyngeal nerve supplying the levator veli palatini muscle. These findings indicate that the motor nerve supply to the levator veli palatini muscle is the glossopharyngeal nerve, and the levator veli palatini muscle is related to the respiratory system, in particular with inspiration in rats.     

Atrial natriuretic factor--its possible role in the pathogenesis and therapy of arterial hypertension

Using radioimmunoassay the authors investigated the plasma concentration of the immunoreactive atrial natriuretic factor (IR-ANF) and its content in the atria of 4-, 8-, 12-, 16-, and 20-weeks-old spontaneously hypertensive rats (SHR), and compared the results with data obtained in normotensive Wistar-Kyoto rats of the same age. With hypertension accelerating in SHR between the 8th and the 20th weeks of life, IR-ANF content in the atrium gradually decreased, and the plasma IR-ANF concentration increased. The decline in IR-ANF was due to its decrease primarily in the left atrium. Long-term (6-day) administration of synthetic ANF to SHR with fully developed hypertension led to normalization of BP. The results do not support the hypothesis that arterial hypertension in SHR is induced by a primary deficiency of ANF. The changes in IR-ANF in the atria and plasma occur rather as an adaptive and regulatory response to increasing BP. Prolonged administration of ANF to SHR had a hypotensive effect. Therapeutic application of ANF in man depends on the development of oral and long-acting analogues.     

Rapid diagnosis of pneumonia in children

Antigen detection techniques are available for the identification of bacterial polysaccharides, viruses, and chlamydia. Viruses and chlamydia are detected by direct immunofluorescence (DFA) or enzyme immunoassay (EIA). Bacterial polysaccharides are detected by latex agglutination or staphylococcal coagglutination of serum or concentrated urine. Most studies have not compared these techniques to the gold standard of lung puncture, so the role of dual infections with bacteria and viruses cannot be adequately determined. The sensitivity of any of these techniques is dependent on the quality of the antisera used. Monoclonal sera are now available for the detection of most viruses and seem to be as sensitive as polyclonal sera. DFA or EIA may offer equal sensitivity but their advantages and disadvantages must be considered by the local diagnostic laboratories. Most DFA and EIA systems have a sensitivity of 90% when compared with viral cultural for the identification of the organism. Agglutination reagents are available commercially for the detection of pneumococcal and Hemophilus influenzae type b polysaccharides. The sensitivity and specificity of each brand should be determined on serum or urine from patients known to have positive blood cultures and those free of disease. The brand chosen should be the one that has reasonable sensitivity and specificity. Rapid diagnostic techniques are helpful if they are used within a clinical context and they are positive. Negative tests do not rule out infection.     

Application of cytopathology in a sensitivity test for anti-tumor agents. I. An experimental study

Although the human tumor clonogenic assay (HTCA) is extremely reliable in determining clinical correlations, it is a complicated process requiring considerable time in order to obtain results. Thus, an experimental study on cytopathologic observation (cytologic assay) and comparative evaluation between it and HTCA were performed in order to establish a more rapid and accurate drug sensitivity test. Materials included Colon 26, a cell line established in our department, malignant effusion and surgical specimens. In carrying out HTCA according to the Hamburger-Salmon method, the cell suspension samples following exposure to anti-tumor agents (MMC, L-PAM, ADM, CDDP) were cultivated in test tubes for 3-8 hours and stained by the Papanicolaou and Giemsa methods. According to Tokita's criteria, when cellular changes showed as nuclear pyknosis and nuclear destruction were found to have increased significantly in comparison with a control group, the cells were judged to be sensitive. Very similar and parallel results were obtained between HTCA and cytologic assay in this study, with a significant correlation. Cytologic assay was proved to be an easy, rapid and accurate method for testing drug sensitivity and its clinical application can be expected in the future.