Nasal immunization of mice with peptide having a cross-neutralization epitope on minor capsid protein L2 of human papillomavirus type 16 elicit systemic and mucosal antibodies.

A common cross-neutralization epitope for human papillomavirus types 6 and 16 (HPV 6 and 16) is present in the region of amino acids (aa) 108-120 of HPV-16 minor capsid protein, L2. We nasally immunized Balb/c mice with a synthetic peptide with the 13 aa HPV 16 L2 sequence, and examined the antibodies elicited. ELISA showed that the immunization induced predominantly IgG and IgA antibodies cross-binding to L1/L2-capsids of HPVs 6, 16, and 18 in sera and in vaginal secretions, respectively. The serum containing the IgG antibody and the vaginal wash containing the IgA antibody neutralized HPV 16 pseudovirions and HPV 11 authentic virions, as shown by surrogate infectivity assays. From their cross-binding activity for HPV 16 and 18, the peptide-induced antibodies can probably cross-neutralize most of the genital HPVs. The peptide-induced neutralizing activity in vaginal wash was comparable to that induced by nasally immunization with HPV 16 L1-capsids. Unlike Balb/c, C57BL/10, which has different MHC class II, did not respond to the peptide immunization, but aa substitutions in the peptide to fulfill the requirement for the C57BL/10 agretope rendered the modified peptides immunogenic. The results provide a basis for development of a peptide vaccine against broad-spectrum of genital HPVs for humans.

Human papillomavirus type 16 minor capsid protein l2 N-terminal region containing a common neutralization epitope binds to the cell surface and enters the cytoplasm.

The first step of papillomavirus infection is believed to be binding of major capsid protein L1 to the cell surface without involvement of minor capsid protein L2, but the viral infectivity can be neutralized either by anti-L1 or anti-L2 antibody. To understand the role of L2 in human papillomavirus (HPV) infection, we examined a segment of HPV type 16 (HPV16) L2, which contains a neutralization epitope common to HPV6, for its involvement in adsorption and penetration of the capsids. Preincubation of monkey COS-1 cells with a synthetic peptide having amino acids (aa) 108 to 120 of HPV16 L2 reduced the susceptibility of COS-1 cells to infection with HPV16 pseudovirions. Confocal microscopy showed that the green fluorescence protein (GFP) fused with the L2 peptide was found to bind to the surface of a HeLa cell, an HPV18-positive human cancer cell line, at 4 degrees C and to enter the cytoplasm after subsequent incubation at 37 degrees C. Flow cytometry showed that fused GFP did not bind to HeLa cells that had been treated with trypsin. Besides COS-1 and HeLa cells, some human and rodent cell lines were detected by flow cytometry to be susceptible to binding with fused GFP, showing a tendency of epithelial cells toward higher susceptibility. Substitutions at aa 108 to 111 inhibited fused GFP from binding to HeLa cells and reduced the infectivity in COS-1 cells of the in vitro-constructed pseudovirions. The results suggest that L2 plays an important role in enhancing HPV infection through interaction between the N-terminal region and a cellular surface protein, facilitating penetration of the virions and determining part of the tropism of HPVs.

Location of KAI1 on the short arm of human chromosome 11 and frequency of allelic loss in advanced human prostate cancer.

BACKGROUND: We recently isolated the KAI1 gene, a metastasis suppressor gene for prostate cancer, from human chromosome region 11p13-cen-containing rat prostate cancer cells. The present study was performed to further locate the region of the KAI1 gene on the short arm of chromosome 11, and to examine whether loss of this region is significant during progression of human prostate cancer. METHODS: The small portion of human chromosome 11 (i.e., 11p13-cen) was reintroduced into highly metastatic rat prostate cancer cells by using microcell-mediated chromosome transfer. Loss of heterozygosity (LOH) at polymorphic microsatellite loci on the human chromosome 11 was examined in human prostate cancer tissues. RESULTS: The minimum region of human chromosome 11 that contained the KAI1 gene was located on the proximal region of 11p11.2 divided by the D11S554 locus. The percentage of LOH or allelic imbalance at the D11S1344 locus, which is located on the same region as the KAI1 locus, in metastasis tissues from autopsy cases who died from metastatic prostate cancer was 70% (7 of 10 informative cases), whereas the percentages in primary tumors from the same cases and from cases with clinically localized prostate cancer were 33% (3 of 9 informative cases) and 8% (1 of 12 informative cases), respectively. CONCLUSIONS: These findings demonstrate a high frequency of LOH or allelic imbalance at the centromeric region of 11p, which contains the KAI1 gene in advanced prostate cancer.

Suppression of invasive ability of highly metastatic rat prostate cancer by introduction of human chromosome 8.

BACKGROUND: Introduction of human chromosome 8 to a highly metastatic subline (AT6.2) from the Dunning R-3327 rat prostate cancer resulted in suppression of metastatic ability of the resultant microcell hybrids (AT6.2-8 clones) [12]. The present study has been performed to clarify which step of metastasis was suppressed in the microcell hybrids. METHODS: Northern blot analysis of E-cadherin and alpha-catenin, in vitro invasion assay, and intra-venous metastasis assay by injection of tumor cells into the lateral tail vein of nude mice were performed. RESULTS: No detectable expressions of either E-cadherin or alpha-catenin were found in either AT6.2 parental or AT6.2-8 microcell hybrid clones. In the invasion assay, invasiveness of AT6.2-8 hybrid clones was less than that of the AT6.2 parental clone. In the intravenous metastasis assay, no significant differences in the number of lung metastases were observed among these cell lines. CONCLUSIONS: Introduction of human chromosome 8 to AT6.2 cells shows suppression of invasiveness and no suppression of cell dissociation or process after entry into blood circulation. This suggests that human chromosome 8 contains suppressor gene(s) for the invasive ability of prostate cancer.

Mapping of metastasis suppressor gene(s) for rat prostate cancer on the short arm of human chromosome 8 by irradiated microcell-mediated chromosome transfer.

Our previous studies demonstrated that human chromosome 8 contains metastasis suppressor gene(s) for rat prostate cancer. However, it is still unknown which portion of human chromosome 8 is associated with suppression of metastatic ability, because all of the clones in which metastatic ability is suppressed contain at least one copy of intact human chromosome 8. In the present study, we used the irradiated microcell-mediated chromosome transfer technique to enrich for specific chromosomal arm deletions of selected chromosomes. The resultant series of human chromosomes 8 with a variety of chromosomal deletions was introduced into highly metastatic Dunning rat prostate cancer cells. All of the resultant microcell hybrids showed reduced metastatic ability. To obtain a smaller size of human chromosome 8 and to locate further the region of metastasis suppressor gene(s), the most reduced size of human chromosome 8 that was generated with the initial irradiated chromosome transfer was retransferred into the Dunning cancer cells without irradiation. The resultant microcell hybrids were analyzed to determine which portion of human chromosome 8 suppressed the metastatic ability of the recipient cells. This analysis demonstrates that the portion of human chromosome 8 containing metastasis suppressor gene(s) for rat prostate cancer cells lies on human chromosome segment 8p21-p12, where frequent allelic losses have been detected in allelotype analyses of human prostate cancer. This suggests that one of the metastasis suppressor genes for rat prostate cancer on human chromosome 8 may also play an important role in the progression of human prostate cancer.

Malignant hyperthermia during epidural anesthesia.

We anesthesized a patient susceptible to malignant hyperthermia (MH) three different times by epidural anesthesia with different types of local anesthetics. His skinned fiber test showed a marked acceleration of calcium (Ca2+)-induced Ca2+ release (CICR). When ester type local anesthetic was used for ankle contracture repair, MH signs appeared following the release of the tourniquet. CICR test is reliable for diagnosing different types of MH.

Metastasis suppressor genes for prostate cancer.

To examine the role of human chromosomes in the development of metastatic prostate cancer, we introduced a copy of human chromosomes into highly metastatic Dunning R-3327 rat prostatic cancer cells by microcell-mediated chromosome transfer. Each microcell hybrid clones containing human chromosomes 8, 10, 11, and 17, respectively, showed decreased ability to metastasize to the lung, without any loss of tumorigenicity. This finding demonstrates that these human chromosomes contain metastasis suppressor genes for prostate cancer. Spontaneous deletion of portions of human chromosomes was observed in human chromosome 10, 11, and 17 studies. In the human chromosome 8 study, irradiated microcell-mediated chromosome transfer was performed to enrich chromosomal arm deletions of human chromosome 8. Relationships between the size of human chromosomes introduced into microcell hybrid clones and the number of lung metastases produced by the clones were analyzed to determine which part of human chromosomes contained metastasis suppressor gene(s) for prostate cancer. Molecular and cytogenetic analyses of microcell hybrid clones demonstrated that metastasis suppressor genes on human chromosomes 8, 10, and 11 were located on 8p23-q12, 10q, 11p13-11.2, respectively. Further analyses are proposed to confirm the potentially useful advantage of this assay system to identify metastasis suppressor gene(s) for prostate cancer.

Localization of metastasis suppressor gene(s) for rat prostatic cancer to the long arm of human chromosome 10.

To examine the role of human chromosome 10 in development of prostatic cancer, we introduced human chromosome 10 into highly metastatic rat prostatic cancer cells by microcell-mediated chromosome transfer. Microcell hybrid cells introduced with human chromosome 10 showed suppression of the metastatic ability to the lung to some extent without any suppression of tumorigenicity, although the tumor growth rate decreased slightly. To minimize the region that contains metastasis suppressive activity, the hybrid cells in metastasis foci of lung were established in culture and reanalyzed for portions of human chromosome 10 retained in the metastasis tissues. Cytogenetic and molecular analyses demonstrated that loss of the region between 10cen and D10S215 on human chromosome arm 10q was related to expression of the metastatic phenotype. These results demonstrate that the region between 10cen and D10S215 on human chromosome arm 10q contains at least one of the metastasis suppressor genes for rat prostatic cancer.

Enhancement of Ca2+-induced Ca2+ release in calpain treated rabbit skinned muscle fibers.

Calpain treatment of rabbit skinned muscle fibers resulted in proteolysis of junctional foot protein or Ca2+ release channel of the sarcoplasmic reticulum. Electrophoretic and immunoblot analyses indicate that calpain cleaves off approximately 130 kDa peptide from the N-terminus. After such treatment, Ca2+ capacity of the sarcoplasmic reticulum remained normal and both Ca2+ and adenine nucleotide dependence of Ca2+-induced Ca2+ release mechanism were retained. However, the Ca2+-activated Ca2+ release rate was increased by two fold after the proteolysis. The results suggest the presence of functional domains in the junctional foot protein, and the N-terminus domain controls the activity of the Ca2+ channel without changing Ca2+ and nucleotide sensitivities.

[Effects of halothane, caffeine and ryanodine on the intracellular calcium store in blood mononuclear cells].

To evaluate the possibility of using blood cells in the screening test for susceptibility to malignant hyperthermia (MH), we examined the effect of halothane and caffeine on cytoplasmic free calcium concentration ([Ca]i) in mononuclear cells. Blood mononuclear cells were isolated from guinea pigs or normal human volunteers, loaded with fura-2 AM and changes in the calcium signal (340nm/380nm ratio) after the application of halothane and/or caffeine were measured. Halothane above 5 mM caused a large increase in [Ca]i, but this increase was mostly abolished by the removal of extra-cellular calcium using EGTA. On the other hand, caffeine caused no observable change in the calcium signal. Pre-treatment with ryanodine did not change the calcium signal brought about by halothane or ionomycin. We conclude from this study that there is no calcium-induced calcium release (CICR) mechanism detectable by this method in blood mononuclear cells. As we consider that the main cause of the typical MH is the abnormality in the CICR mechanism, it seems difficult to screen MH susceptibility by using blood mononuclear cells. Further studies will be necessary using MH susceptible swines or patients.

[Coronary artery spasm under thoracic epidural anesthesia].

Three paroxysmal episodes of ST-segment elevation in lead II of ECG were observed during bullectomy and chest closing under epidural anesthesia supplemented with enflurane in compressed air in a patient who had history of variant angina with 50% obstruction of right coronary artery. The first and the third episodes were followed by ventricular tachycardia, complete A-V block and hypotension. These attacks were preceded by decreases in heart rate and blood pressure. It was suspected that coronary artery spasm developed with increased vagal tone under thoracic epidural block. The first and the second attacks were successfully treated with intravenous injection of nitroglycerin and lidocaine. The third attack needed additional treatments which included intravenous administrations of atropine, epinephrine, isoproterenol and phenylephrine and direct heart massage through the thoracic incision. Postoperative serial examinations of ECG showed inverted T in lead V1-V4, and serum enzymes (GOT, GPT, LDH, CPK, CPK-MB) were elevated. However ratio of CPK-MB to total CPK was only 1.5%. The patient was discharged two weeks after the operation with normal ECG and serum enzymes. It is speculated that coronary artery spasm was induced by hypotension and vagal stimulation under epidural anesthesia which blocks cardiac sympathetic nerves.

Epidural ketamine for postoperative pain relief after gynecologic operations: a double-blind study and comparison with epidural morphine.

This double-blind study evaluates whether ketamine given epidurally is effective for postoperative pain relief, and compares the effects of epidural ketamine with those of epidural morphine. Sixty-eight patients undergoing abdominal gynecologic surgery were randomly assigned into six groups (control; ketamine 4, 6, and 8 mg in saline; 6 mg in 10% glucose; morphine 3 mg). All patients were anesthetized with thiopental, nitrous oxide, and enflurane, and drugs were administered epidurally at the end of the operation. The duration of analgesia in the ketamine groups did not differ from that in control patients and the difference in diluent had no observable effects. Significantly, none of the patients in the morphine group needed additional analgesics within 24 hr, whereas 85% in the other five groups did. We conclude that ketamine administered epidurally is inadequate for postoperative pain relief after gynecologic operations.