Protective efficacy of rotavirus 2/6-virus-like particles combined with CT-E29H, a detoxified cholera toxin adjuvant.

Identifying a safe and efficacious mucosal adjuvant is crucial for the development of subunit vaccines against rotavirus and other mucosal pathogens. Moreover, recognition of determinants of protective immunity to rotavirus infection is essential to the design of the means to prevent or control this viral gastrointestinal disease. We have studied the kinetics of systemic and mucosal antibody responses elicited upon mucosal immunization of mice with rotavirus recombinant virus-like particles (rVLPs) alone or combined with a detoxified version of cholera toxin, CT-E29H. CT-E29H has been shown to maintain the adjuvant effect of parental cholera holotoxin. Both inbred BALB/c and outbred CD-1 mice were immunized with rotavirus VP2/6-rVLPs (2/6-VLPs) combined with CT-E29H, orally or intranasally (i.n.), and the comparative efficacy of different formulations was then determined. Rotavirus-specific serum and fecal IgA, IgM, and IgG antibodies were determined by enzyme-linked immunoadsorbent assay (ELISA) weekly (or every other week) following vaccination. Animals then were challenged with a murine rotavirus strain, EDIM. The degree to which vaccinated animals were protected from the wild-type rotavirus challenge was reflected in the levels of viral antigen shed in stools (percent reduction in antigen shedding, PRAS). BALB/c mice immunized by either route produced rotavirus-specific serum IgA, IgM and IgG, as well as fecal IgA and IgG, but not IgM; however, the intranasal immunization induced stronger systemic IgG and IgM responses than did oral immunization. Similar levels of prechallenge rotavirus-specific fecal and serum IgA were detected in both the orally and the i.n. immunized groups. Two immunizations with 2-6VLPs and CT-E29H were sufficient to protect BALB/c mice, regardless of the route of administration. PRAS was 99.6, 98.8, and 98.8% for oral, i.n. and the oral + i.n. groups, respectively; in contrast vaccination with 2/6-VLPs alone was not protective (PRAS = 39%), indicating the critical role of CT-E29H in inducing protective levels of immune responses. Two of four outbred CD-1 mice that were immunized orally with 2/6-VLPs-CT-E29H showed no humoral responses (PRAS, 65%), but four of four i.n. immunized CD-1 mice displayed humoral responses (PRAS, 97.9%). Serum anti-VP6 and VP2 antibodies were detected in all immunoresponsive mice. The combined results in two strains of mice indicate that CTE29H is an effective mucosal adjuvant capable of inducing protective immune responses and suggest that intranasal administration is the preferred route of immunization.

Clinical impact of second harmonic imaging and left heart contrast in echocardiographic stress testing.

Second harmonic imaging and left heart contrast agents are recent echocardiographic advancements that enhance the assessment of wall motion. Because little information exists concerning their clinical impact on echocardiographic stress testing in daily practice, this was determined for 9-month periods before (1997) and after (1998) their introduction. Harmonic imaging was used in all patients after its introduction. A second generation intravenous left heart contrast agent (Optison) was used at the discretion of the sonographer and physician team. Both exercise and dobutamine stress tests were included. At the time of study interpretation, diagnostic confidence was assigned as high, medium, or low. For all patients who underwent coronary angiography < or = 6 months after stress testing, the diagnostic accuracy was determined (true positive plus true negative/total studies). There were 574 studies before and 746 studies after implementation. Optison was used in 28% of the harmonic imaging studies. Study cancellations due to uninterpretable images fell from 6.4% to 1.2% (p <0.001) despite a more obese population completing testing (body mass index: 29 +/- 7 to 31 +/- 8 kg/m2, p = 0.02), whereas high diagnostic confidence increased from 55% to 64% (p <0.001). For the 7% of patients who underwent cardiac catheterization, the diagnostic accuracy remained unchanged (74 vs 73%) although a prior negative stress test was less common (40% to 20% p = 0.04). Thus, these new technologies had a favorable clinical impact.

Protective effect of rotavirus VP6-specific IgA monoclonal antibodies that lack neutralizing activity.

Rotaviruses are the leading cause of severe gastroenteritis and dehydrating diarrhea in young children and animals worldwide. A murine model and "backpack tumor" transplantation were used to determine the protective effect of antibodies against VP4(an outer capsid viral protein) and VP6(a major inner capsid viral protein). Only two non-neutralizing immunoglobulin A (IgA) antibodies to VP6 were capable of preventing primary and resolving chronic murine rotavirus infections. These antibodies were not active, however, when presented directly to the luminal side of the intestinal tract. These findings support the hypothesis that in vivo intracellular viral inactivation by secretory IgA during transcytosis is a mechanism of host defense against rotavirus infection.

Introduction of a fast and sensitive fluorescent in situ hybridization method for single-copy detection of human papillomavirus (HPV) genome.

At present, in situ hybridization (ISH) is the only method for detection of specific genes in morphologically intact cells or tissue. We have developed a highly sensitive and quantitative fluorescence-based in situ hybridization (FISH) technique that can detect as few as one to five copies of the integrated human papillomavirus (HPV) type 16 genome in cervical cell lines, using digoxygenin tail-labeled oligonucleotides (Method 1). The entire procedure can be carried out in 4.5 hr through the elimination of some of the steps routinely used in other ISH protocols. We also compared the sensitivity of this new FISH method (Method 1) to four other FISH techniques: digoxigenin-labeled DNA probe (Method 2); fluorescein-15-d-ATP-labeled oligonucleotides (Method 3); fluorescein 15-d-ATP labeled DNA probe (Method 4); and biotin-DNA-labeled probe (Method 5), for their ability to detect HPV DNA in the HPV-positive human cervical cell lines CaSki (500 copies) and SiHa (1-5 copies), but not in C33-A and HT-3, which do not contain any copies of HPV. Our results indicate that Method 1 is more sensitive than the other methods employed. Method 1 was the only method that could reliably detect an HPV-16 genome in all SiHa cells. Our data suggest that the Method 1 FISH technique is highly sensitive and may therefore be of general use for detection and quantitation of a variety of viral genomes (including HIV), oncogenes, and drug-resistant genes, in a variety of morphologically intact cells and tissues.

Detection of human papillomavirus type 16/18 DNA in cervicovaginal cells by fluorescence based in situ hybridization and automated image cytometry.

Automatic fluorescence image cytometry (AFIC) is a fast, sensitive, and reliable approach for screening slide-based clinical specimens. In this study, we applied AFIC to identify cancer-associated human papillomavirus (HPV) genotypes 16 and 18 in individual cells of cervical smears using a sensitive fluorescence based in situ hybridization (FISH) assay. HPV sequences were labeled by FISH and the cells imaged using an epi-fluorescence microscope coupled to a low-light color CCD camera. Before application to clinical specimens, AFIC was assessed using fluorescent calibration beads and cervical cancer cell lines containing known numbers of integrated HPV genomes per nucleus. Assessment showed that our AFIC had a linear response, was quantitatively accurate, and had the sensitivity to detect one HPV genome per nucleus. After acquisition of images, computer algorithms identified every cell nucleus (via a fluorescent DNA counterstain) and quantified the FISH signal per nucleus. AFIC was employed to screen 27 patient specimens for HPV 16/18, of which 12 were positive. The HPV status of the specimens positively correlated with the pathological diagnosis, and since AFIC automatically and correctly located every cell, it was possible to directly compare morphology and HPV status in the same cell. In conclusion, the combination of FISH and AFIC is a sensitive and quantitative method to detect high risk HPV sequences in cervical smears.

Expression of metalloproteinase genes in human prostate cancer.

Twenty-five surgical specimens of malignant human prostate, 3 lymph nodes with metastatic prostate carcinoma, 11 normal human prostates, as well as 3 human prostate cell lines (DU-145, PC3 and LNCaP) were examined for the expression of the human matrix metalloproteinase-7 gene (MMP-7) from the human collagenase family (originally called PUMP-1 for putative metalloproteinase-1) [Quantin et al. (1989) Biochemistry 28:5327-5334; Muller et al. (1988) Biochem J 253:187-192; Matrisian and Bowden (1990) Semin Cancer Biol 1:107-115]. Northern blots were prepared using total RNA extracted from 18 prostate adenocarcinomas, 2 lymph nodes with metastatic prostate carcinoma and 11 normal human prostates. When the northern blots were hybridized with a 32P-labeled MMP-7 cDNA probe, a 1.2-kb mRNA was detected in 14 out of 18 prostate adenocarcinomas, 1 out of 2 metastatic lymph nodes, and 3 out of 11 normal prostates. The 3 human prostate cell lines did not show any evidence of the MMP-7 transcript. In situ hybridization was conducted to localize the MMP-7 mRNA to individual cells using a 35S-labeled MMP-7 cRNA. In situ hybridization was carried out on snap-frozen tissue sections of 7 prostate adenocarcinomas and 3 lymph nodes containing metastatic prostate adenocarcinoma using the same tissues previously probed by northern analysis as well as new samples. In situ hybridization revealed that the MMP-7 gene was expressed in the epithelial cells of primary prostate adenocarcinoma as well as in invasive and metastatic cells. MMP-7 expression was also seen focally in some dysplastic glands but not in stroma. Additional northern blot analysis was performed using probes to human type-IV collagenase, type-I collagenase and stromelysin I in human prostate adenocarcinoma as well as normal prostate tissue. Our results indicated that 6 out of 10 adenocarcinoma samples and none of the 4 normal samples were positive for type-IV collagenase transcripts. Tissue samples were also examined for the expression of type-I collagenase (9 adenocarcinomas and 4 normal) and stromelysin I (13 adenocarcinomas) by northern analysis. None of the tissues was found to express the transcripts of interest at detectable levels. These data suggest that certain metalloproteinases are present in prostatic adenocarcinoma and may play a role in invasion and metastasis.

Generation of halothane-induced immune response in a guinea pig model of halothane hepatitis.

A guinea pig model of halothane hepatitis was used to explore the humoral immune response induced by multiple halothane exposures and the potential role this response might play in contributing to liver damage. Three different strains of guinea pigs (Strain 2, Amana, and Hartley) were exposed to 1% halothane under either 21 or 80% oxygen for 4 hr at 2-week intervals. In each strain, halothane induced the appearance of an antibody cross-reactive with trifluoroacetylated guinea pig serum albumin (TFA-GSA). Three of six Strain 2 guinea pigs demonstrated an association between antibody titer and serum glutamate pyruvate transaminase levels. However, the possible cause and effect relationship between these two factors requires more investigation. Hartley guinea pigs had a 4- to 11-fold higher level of anti-TFA antibody than the other two strains because of either a "higher responder" genetic background or exposure conditions that favored oxidative metabolism of halothane. Immunization of Amana guinea pigs with TFA-GSA evoked a specific anti-TFA antibody response. However, the presence of this antibody before halothane exposure did not potentiate the transient liver damage induced by exposure. Thus, these results demonstrate that in guinea pigs multiple exposures to halothane induce the formation of an antibody that recognizes a reactive intermediate of halothane formed during the anesthetic's metabolism.