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Purpose: Histopathology is the standard approach for tissue diagnostics and centerpiece of pathology. Although the current system provides prognostic information, there is need for molecular markers that enhance diagnosis and better predict clinical prognosis. The ability to localize disease-specific molecular changes in biopsy tissue would help improve critical pathology decision making. Direct profiling of proteins from tissue using matrix-assisted laser desorption/ionization imaging mass spectrometry has the potential to supplement morphology with underlying molecular detail.Experimental Design: A discovery set of 11 prostate cancer (PCa)–containing and 10 benign prostate tissue sections was evaluated for protein expression differences. A separate validation set of 54 tissue sections (23 PCa and 31 benign) was used to verify the results. Cryosectioning was done to yield tissue sections analyzed by a pathologist to determine tissue morphology and mirror sections for imaging mass spectrometry. Spectra were acquired and the intensity of signals was plotted as a function of the location within the tissue.Results: An expression profile was found that discriminates between PCa and normal tissue. The overexpression of a single ion at m/z 4,355 was able to discriminate cancer from uninvolved tissue. Tandem mass spectrometry identified this marker as a fragment of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 2 (MEKK2). The ability of MEKK2 to discriminate tumor from normal cells was orthogonally confirmed.Conclusions: This study highlights the potential of this approach to uncover molecular detail that can be correlated with pathology decision making. In addition, the identification of MEKK2 shows the ability to discover proteins of relevance to PCa biology. (Clin Cancer Res 2009;15(17):5541–51)

Welcome to the Reimagining International Development issue of the journal. It is inspired by an online Conference titled Healing Solidarity: Re-imagining International Development, the brainchild o...

Diabetic non-healing wounds are a major clinical problem. The mechanisms leading to poor wound healing in diabetes are multifactorial but unresolved inflammation may be a major contributing factor. The complement system (CS) is the most potent inflammatory cascade in humans and contributes to poor wound healing in animal models. Signal transducer and activator of transcription 4 (STAT4) is a transcription factor expressed in immune and adipose cells and contributes to upregulation of some inflammatory chemokines and cytokines. Persistent CS and STAT4 expression in diabetic wounds may thus contribute to chronic inflammation and delayed healing. The purpose of this study was to characterize CS and STAT4 in early diabetic wounds using db/db mice as a diabetic skin wound model. The CS was found to be activated early in the diabetic wounds as demonstrated by increased anaphylatoxin C5a in wound fluid and C3-fragment deposition by immunostaining. These changes were associated with a 76% increase in nucleated cells in the wounds of db/db mice vs. controls. The novel classical CS inhibitor, Peptide Inhibitor of Complement C1 (PIC1) reduced inflammation when added directly or saturated in an acellular skin scaffold, as reflected by reduced CS components and leukocyte infiltration. A significant increase in expression of STAT4 and the downstream macrophage chemokine CCL2 and its receptor CCR2 were also found in the early wounds of db/db mice compared to non-diabetic controls. These studies provide evidence for two new promising targets to reduce unresolved inflammation and to improve healing of diabetic skin wounds.

The prostate gland secretes many proteins in a prostatic fluid that combines with seminal vesicle derived fluids to promote sperm activation and function. Proximal fluids of the prostate that can be collected clinically are seminal plasma and expressed-prostatic secretion (EPS) fluids. EPS represents the fluid being secreted by the prostate following a digital rectal prostate massage, which in turn can be collected in voided urine post-exam. This collection is not disruptive to a standard urological exam, and it can be repeatedly collected from men across all prostatic disease states. A direct EPS fluid can also be collected under anesthesia prior to prostatectomy. While multiple genetic assays for prostate cancer detection are being developed for the shed epithelial cell fraction of EPS urines, the remaining fluid that contains many prostate-derived proteins has been minimally characterized. Approaches to optimization and standardization of EPS collection consistent with current urological exam and surgical practices are described, and initial proteomic and glycomic evaluations of the of EPS fluid are summarized for prostate specific antigen and prostatic acid phosphatase. Continued characterization of the prostate specific protein components of EPS urine combined with optimization of clinical collection procedures should facilitate discovery of new biomarkers for prostate cancer.

Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) are glycoproteins secreted by prostate epithelial cells, and have a long clinical history of use as serum biomarkers of prostate cancers. These two proteins are present at significantly higher concentrations in seminal plasma, making this proximal fluid of the prostate a good source for purifying enough protein for characterization of prostate disease associated changes in glycan structures. With the use of seminal fluid samples representative of normal control, benign prostatic disease and prostate cancers, PAP and PSA were enriched by thiophilic absorption chromatography. Released N-linked glycan constituents from both proteins were analyzed by a combination of normal phase HPLC and MALDI-TOF spectrometry. For PSA, 40 putative glycoforms were determined, and 21 glycoforms were determined for PAP. PAP glycans were further analyzed with a hybrid triple quadrupole/linear ion trap mass spectrometer to assign specific glycoform classes to each of the three N-linked sites. The glycans identified in these studies will allow for more defined targeting of prostate disease-specific changes for PAP, PSA and other secreted prostatic glycoproteins.

We present a method of data reduction using a wavelet transform in discriminant analysis when the number of variables is much greater than the number of observations. The method is illustrated with a prostate cancer study, where the sample size is 248, and the number of variables is 48,538 (generated using the ProteinChip technology). Using a discrete wavelet transform, the 48,538 data points are represented by 1271 wavelet coefficients. Information criteria identified 11 of the 1271 wavelet coefficients with the highest discriminatory power. The linear classifier with the 11 wavelet coefficients detected prostate cancer in a separate test set with a sensitivity of 97% and specificity of 100%.

Induction of CD8 + cytotoxic T cells is considered one of the important correlates for the protective efficacy of candidate human immunodeficiency virus type 1 (HIV-1) vaccines. To induce CD8 + cytotoxic T lymphocytes (CTLs) along with neutralizing antibody and CD4 + T cell help, a live canarypox virus construct expressing gp120, transmembrane gp41, the gag and protease genes, and sequences containing CTL epitopes in nef and pol was given simultaneously with, or followed by, rgp120 SF2. CD8 + CTLs were detected in 61% of volunteers at some time during the trial. Three to 6 months after the last immunization, the gene-specific responses were gag, 26/81; env, 17/77; nef, 12/77; and pol, 3/16. Simultaneous immunization with the canarypox vector and the subunit, beginning with the initial immunization, resulted in earlier antibody responses. In summary, a strategy of immunization with a canarypox vector expressing multiple genes of HIV-1 given with gp120 results in durable CD8 + CTL responses to a broad range of epitopes.

A safety and immunogenicity trial was conducted in vaccinia-immune and vaccinia-naive human immunodeficiency virus (HIV)-uninfected adults who were randomized to receive 10(6) or 10(7) TCID50 of canarypox (ALVAC) vector expressing HIV-1MN gp160 or 10(5.5) TCID50 of ALVAC-rabies virus glycoprotein control at 0 and 1 or 2 months and ALVAC-gp160 or 50 microg of HIV-1SF2 recombinant (r) gp120 in microfluidized emulsion at 9 and 12 months; others received rgp120 at 0, 1, 6, and 12 months. All vaccines were well-tolerated. Neither vaccinia-immune status before vaccination nor ALVAC dose affected HIV immune responses. HIV-1MN and HIV-1SF2 neutralizing antibodies were detected more often (100%) in ALVAC-gp160/rgp120 recipients than in recipients of ALVAC-gp160 (

A live, cold-passaged ( cp ) candidate vaccine virus, designated respiratory syncytial virus (RSV) B1 cp -52/2B5 ( cp -52), replicated efficiently in Vero cells, but was found to be overattenuated for RSV-seronegative infants and children. Sequence analysis of reverse-transcription–PCR-amplified fragments of this mutant revealed a large deletion spanning most of the coding sequences for the small hydrophobic (SH) and attachment (G) proteins. Northern blot analysis of cp -52 detected multiple unique read-through mRNAs containing SH and G sequences, consistent with a deletion mutation spanning the SH:G gene junction. Immunological studies confirmed that an intact G glycoprotein was not produced by the cp -52 virus. Nonetheless, cp -52 was infectious and replicated to high titer in tissue culture despite the absence of the viral surface SH and G glycoproteins. Thus, our characterization of this negative-strand RNA virus identified a novel replication-competent deletion mutant lacking two of its three surface glycoproteins. The requirement of SH and G for efficient replication in vivo suggests that selective deletion of one or both of these RSV genes may provide an alternative or additive strategy for developing an optimally attenuated vaccine candidate.

BACKGROUND Tumorigenesis and progression to metastatic disease are accompanied by changes in the expression of cell adhesion molecules (CAMs). Normally expressed CAMs, such as E-cadherin, are lost, while others, i.e., ICAM-1, VCAM-1, NCAM, and E-selectin, are altered and overexpressed in progressive disease and metastases. Abnormal levels of these latter CAMs have been observed in melanoma and carcinomas of the colon and breast, and NCAM is overexpressed in small-cell lung carcinoma (SCLC). The objective of this study was to determine if serum levels of ICAM-1, VCAM-1, NCAM, and E-selectin could differentiate patients with benign prostate hypertrophy (BPH) from those with prostate carcinoma (CaP) and identify prostate cancers with high potential for progression to metastatic disease. METHODS Serum levels of these CAMs were determined by ELISA in serum from normal males and females and from patients with BPH and CaP before and after treatment. Sera from patients with breast carcinoma, colon carcinoma, melanoma, and small-cell lung carcinoma were also evaluated, as soluble CAMs have been reported to be elevated in these cancer patients. RESULTS ICAM-1 levels were elevated in sera from patients with breast carcinoma (P = 0.0004) and melanoma (P = 0.0001). VCAM-1 levels were elevated in sera from patients with colon carcinoma (P = 0.0001). NCAM levels were elevated in the sera of patients with SCLC (P = 0.0001). Normal levels of ICAM-1, E-selectin, and NCAM were found in both BPH and pretreatment CaP patients. Median NCAM levels in hormone-refractive CaP patients were significantly greater than in BPH (P = 0.0005) and CaP patients with pathologically determined organ-confined (P = 0.0014) or nonorgan-confined disease (P = 0.0385). VCAM-1 levels were significantly elevated in both BPH patients (P = 0.0002) and CaP patients (P = 0.0002) when compared with levels for normal age-matched donors. None of the CAMs were found to offer an advantage over prostatic-specific antigen (PSA) for monitoring CaP patients following definitive radiotherapy, radical prostatectomy, or hormonal therapy. CONCLUSIONS The results of this study indicate that serum ICAM-1, VCAM-1, NCAM, and E-selectin are not clinically useful biomarkers for differentiating CaP from BPH, for predicting progression, for identifying metastatic potential, or for monitoring treatment. Prostate 32:214–220, 1997. © 1997 Wiley-Liss, Inc.

Abstract α 1 -Adrenergic receptor contraction of vascular smooth muscle is augmented by increases in angiotensin II and also in several forms of hypertension. Whether angiotensin directly modulates α 1 -adrenoceptor subtype expression to contribute to this effect is unknown. In a previous study, we demonstrated that increased mechanical load (pressure) per se does not alter expression of α 1B - and α 1D -adrenoceptors in rat aortic smooth muscle in cell culture, in vitro or in vivo. However, findings in aortic coarctation hypertension suggested that a humoral factor, possibly angiotensin, selectively reduces α 1B -adrenoceptors and that increased mechanical load opposes this decrease. The present study examined this hypothesis by determining the effect of angiotensin alone and in the presence of mechanical loading on the expression of α 1D - and α 1B -adrenergic receptor mRNAs and α 1 -receptor density in cultured aortic smooth muscle cells. α 1D mRNA content, per smooth muscle cell, concentration-dependently decreased after 3 hours of exposure to 0.3 nmol/L to 1 μmol/L angiotensin but by 24 hours had returned to control levels. In contrast, α 1B mRNA concentration-dependently declined at a later time (24 hours) and remained decreased at 48 hours to 27±6% of control with 1 μmol/L angiotensin. Angiotensin also decreased α 1 -adrenoceptor density in a dose-dependent manner. Angiotensin had no effect on cell number in these confluent, quiescent cells but did increase cell protein and total RNA. This cellular hypertrophy and the decreases in α 1 -adrenoceptor mRNAs were blocked by the angiotensin type 1 receptor antagonist losartan. Cyclic mechanical loading of smooth muscle cells opposed the angiotensin-mediated hypertrophy and decrease in α 1B mRNA expression and α 1 -adrenergic receptor density. These data suggest that angiotensin and intravascular pressure interact to affect cell growth and expression of α 1B -adrenergic receptors by vascular smooth muscle.

Background: Residency programs are responsible for adequately assessing the skills of housestaff. Purpose: To determine the feasibility and reliability of nurses evaluating clinical competence of Internal Medicine (IM) housestaff. Methods: IM housestaff were evaluated by nurses, faculty, and the program director. Composite scores were calculated for skills classified as clinical/technical or interpersonal. Generalizability analysis was used to determine the number of items required and the nmber of evaluation forms to be completed to achieve acceptable reliability. Results: IM housestaff were rated highly by all types of evaluators. Ratings by nurses and faculty were global. There was a low correlation between ratings by nurses and faculty of clinical/technical skills. To achieve an acceptable reliability (r = .80) on clinical/technical skills 5 to 10 faculty were needed to complete 1 to 2 questions, whereas 20 to 30 nurses were required to complete 1 to 2 questions to assess clinical/technical or interpe...

OBJECTIVE: To compare in adults more than 50 years old the tolerability and immunogenicity of vaccination with recombinant hepatitis B surface antigen (HBs) compared with vaccination with recombinant hepatitis B protein PreS2+S, and to investigate the safety and immunogenicity of a fourth vaccine dose in poor and non-responders. DESIGN: Randomized, double-blind prospective study. SETTING: General clinical research center for outpatient evaluation and vaccination. SUBJECTS: Adults older than age 50 who were in general good health and with no known risk factors for acquiring or serologic evidence of hepatitis B virus infection. INTERVENTION: Subjects were randomized to receive 10 mcg HBs (Recombivax®, Merck, Sharp and Dohme), 12 mcg PreS2+S, or 24 mcg PreS2+S vaccine at 0, 1, and 6 months. Poor and non-responders (anti-Hbs < 10 mIU/mL at month 9 and/or 12) were encouraged to receive a fourth vaccine injection. MEASUREMENTS: Diary records of temperature and local and systemic reactions following each vaccination were maintained by all subjects. Anti-HBs levels were measured by radioimmunoassay before the first injection, at 1, 2, 3, 6, 7, 9, and 12 months after for all subjects, and 1 month after the fourth injection for the group of poor and non-responders. MAIN RESULTS: Twenty men and nine women (mean age ± SD, 66 ± 8.0 years) were enrolled. Ten subjects received HBs vaccine, nine received 12 mcg PreS2+S vaccine, and 10 received 24 mcg PreS2 + S vaccine. One subject in the HBs group dropped out, and data were analyzed for the remaining 28 subjects. There were no differences in rates of side effects reported by each of the three groups. Overall, minor local adverse reactions occurred in 12 (40%) after at least one of the first three vaccinations. Systemic side effects occurred in five (17%) after the first vaccination, in one after the second, but in none after the third. The 24-mcg PreS2+S vaccine was not more immunogenic than the HBs vaccine, and the 12-mcg PreS2+S vaccine was judged inadequate. Nineteen of 22 (86%) poor and non-responders received a fourth vaccination. Minor local adverse reactions were reported by six (32%), and none reported a systemic side effect. For the 12 subjects receiving a fourth injection of HBs or 24 mcg PreS2 + S vaccine, the proportion of responders 1 month following the fourth injection was greater than for 1 month following the third injection (11 of 12 [92%] versus 12 of 19 [63%], respectively, P < .05). CONCLUSION: For adults more than 50 years of age who have low anti-HBs levels after three vaccine injections, a fourth injection is well tolerated and results in improved immunogenic response.

A safe and effective influenza vaccine is needed to prevent serious influenza illness in infants younger than 6 months of age. The purpose of this study was to determine whether two doses of the cold-adapted (ca) influenza A reassortant vaccine would be safe and immunogenic in this age group. In the first part of this study, infants received two doses of 105 or 108 50% tissue culture-infectious dose (TCID50) of the ca influenza vaccine separately from routine immunizations. In the second part of this study two 106 TCID50 doses of the ca influenza vaccine were given with routine immunizations at 2 and 4 or 2 and 6 months of age. The ca influenza vaccine was well-tolerated by participants in both parts of this study. Two doses of the ca influenza vaccine were immunogenic in infants who received them separately from routine immunizations; 83% of vaccinees developed protective titers of serum hemagglutination-inhibition (HAI) antibody. In contrast, when the ca vaccine was administered with routine immunizations, protective HAI antibody titers were induced in only 20% of those immunized at 2 and 4 months of age and 50% of those immunized at 2 and 6 months of age. There were no statistically significant associations between HAI antibody response to ca influenza vaccination and dose schedule, presence of passively acquired maternal HAI antibody, ethnic group or breast-feeding status. Young age at the time of first immunization, however, appeared to correlate with decreased response to the hemagglutinin antigen of the influenza A virus. Further studies are needed to determine whether a larger dose of vaccine (107 TCID50) could be used safely to improve the serum antibody response to influenza in very young infants.

Healthy adult volunteers were immunized by parenteral or oral routes with trivalent inactivated influenza vaccine (A/Chile/1/83 (H1N1), A/Mississippi/1/85 (H3N2), and B/Ann Arbor/1/86), or intranasally with live attenuated, cold-adapted influenza type A/Texas/1/85 (H1N1) reassortant virus. In all volunteers, cells spontaneously secreting IgA, IgG or IgM antibodies specific to influenza virus were detected in peripheral blood on days 6-13 after immunization, and specific IgA, IgG and IgM antibodies to influenza vaccine were measured in sera and external secretions (saliva and nasal lavage). Following systemic immunization, a raise in specific antibodies of all isotypes was observed in sera beginning on day 13. Although small variations in IgA and IgM antibodies in saliva and nasal lavages were detected, antigen-specific IgG significantly increased between days 13 and 27. Intranasal administration of attenuated virus induced IgA and IgG antibodies in serum as well as in secretions. Serum antibodies were not substantially influenced by oral immunization, only a small increase in all isotypes was observed in volunteers' sera 21 days after ingestion of vaccine. However, in secretions, antigen-specific IgA and IgG responses were detected one week after immunization and reached a peak response on day 20. These studies show that different routes of immunization can be effective for the induction of specific antibodies, and support the concept of the common mucosal immune system in humans by demonstrating that the oral or intranasal administration of antigen-induced specific antibodies of IgA isotype in external secretions, preceded by the transient appearance in peripheral blood of specific antibody-producing cells.

We examined the safety and immunogenicity of a baculovirus-derived recombinant HIV-1 envelope glycoprotein vaccine candidate, rgp160 (VaxSyn; MicroGeneSys, Meriden, CT), administered at doses of 160 or 640 micrograms to 56 healthy, HIV-1-seronegative adults, in a randomized, double-blind, placebo-controlled study. Immunizations were given intramuscularly at 0, 1, 6, and 12 months. Both doses were generally well tolerated, although self-limited local reactions were frequent. No other clinical or laboratory toxicities were noted, and no effects on CD4 or CD8 lymphocyte counts or percentages were noted. Serum antibody responses to HIV proteins were detected by Western blot (WB) in 19 of 20 and in 19 of 19 recipients of four doses of 160 and 640 micrograms, respectively. Western blot responses developed more rapidly in the 640-micrograms group. High rates of EIA antibody responses to HIV-1 lysate were also present in both groups, and developed more rapidly in the 640-micrograms group. Enzyme immunoassay antibody responses to the immunogen (rgp160) were also frequent, but were infrequent to V3 to gp41 peptides. Neutralizing antibodies against the homologous HIV-1 LAI isolate were seen in 3 of 20 subjects (GMT = 11) who received four doses of 160 micrograms, and in 10 of 19 subjects who received four doses of 640 micrograms (GMT = 32). Fusion inhibiting antibody was not detected. CD4 blocking activity was seen in 3 of 19 subjects who received four doses of 640 micrograms. Complement-mediated antibody-dependent enhancement was found in sera from 11 of 19 volunteers in the 640-micrograms group. Lymphocyte proliferative responses to the immunogen were detected in 4 of 4 subjects tested, but no cytotoxic T cell activity was noted in 11 subjects. Administration of the 640-micrograms dose of this rgp160 vaccine candidate relative to the lower doses was associated with increased immunogenicity, including higher rates of homologous neutralizing antibody responses, although at low titer.

SUMMARY The purpose of this randomized, double-blind study was to test the safely and immunogenicity of an HIV-ILAI recombinant gp160 (rgp160) vaccine in healthy, uninfected volunteers using accelerated dosing schedules. Thirty volunteers were randomly assigned lo receive 50μg doses of rgp160 in one of two immunization schedules. Group 1 received rgp160 at times 0, 1, 2 and 5 months: and group 2 received rgp160 at times 0, 1, 2, 3 and 4 months. The vaccine was safe and stimulated high levels of HIV-1 envelope-specific binding antibody and T cell memory. There was a trend (P < 0.10) suggesting neutralizing antibodies were better induced by the regimen incorporating a rest period before the final immunization in group 1 volunteers. Both accelerated immunization schedules induced immune responses at levels similar to or better than those achieved by four rgp160 vaccine injections given over 12-18 months in other studies.

OBJECTIVE To assess similarities and differences in antibody responses to recombinant (r) HIV-1IIIB gp120 in chimpanzees, previously protected from HIV-1 infection, and human volunteers immunized in connection with a Phase I clinical trial. METHODS Frozen sera from humans immunized with rgp120 from HIV-1IIIB and chimpanzees immunized with the same antigen or recombinant soluble gp160 were compared in a variety of serologic assays. RESULTS The magnitude of the antibody response to gp120 was similar in both species; however, the half-life of the antibody response to rgp120 was approximately 4.5 times longer in humans (9 weeks) than in chimpanzees (2 weeks). Antibodies to gp120 in both species were broadly cross-reactive with gp120 from diverse isolates of HIV-1 and were effective in blocking the binding of gp120 to CD4. Antibody binding to native gp120 was greater than to denatured gp120 in both species. Antibody responses to the principal neutralizing determinant (V3 domain) and virus neutralization titers were approximately 10-fold lower in humans than chimpanzees. The relative avidity of antibody binding to gp120 was higher in the sera from the immunized chimpanzees than in the immunized humans. CONCLUSIONS While the antibody responses to rgp120 elicited in man and chimpanzees were in many ways similar, significant differences did occur. Predictions made on the basis of chimpanzee immunogenicity studies over-estimated the potency of the virus neutralizing titers and under-estimated the duration of the antibody response achieved in humans.

Recombinant gp160 derived from human immunodeficiency virus type 1 (HIV-1)IIIB and produced in mammalian tissue culture cells using a vaccinia virus expression system (rgp160-mam) was evaluated as a vaccine in combination with alum and deoxycholate adjuvant. Sixty low-risk, uninfected subjects received 12.5 micrograms, 50.0 micrograms, or adjuvant control at 0, 1, 6, and 12 months in a randomized, double-blind dose-escalation study. A single injection of 200 micrograms of vaccine was given at 18 months in an open study to 9 vaccines who had received 50 micrograms. The vaccine was safe. Six of 16 subjects receiving 50 micrograms developed neutralizing antibody to HIV-1IIIB. Seven of the 9 boosted with 200 micrograms of vaccine at 18 months developed neutralizing antibodies. Lymphocyte proliferation to rgp160-mam and baculovirus-derived rgp160 and rgp120 was induced in both groups (12.5 and 50.0 micrograms) and appeared after the first dose. Further studies with higher doses of rgp160-mam and vaccines derived from other strains of HIV-1 are warranted.

The development of an effective vaccine against human immunodeficiency virus type 1 (HIV-1) may require immunization protocols that elicit cytolytic T lymphocytes (CTL) in addition to neutralizing antibodies. This report demonstrates that vaccination of 4 HIV-1-seronegative volunteers with a recombinant HIV-1 gp160 vaccine produced in mammalian cells elicited a CTL response in 3. The observed CTL activity was not mediated by classic CD8+ CTL but rather by cells of the CD4+ phenotype. The level of CTL activity varied over time, did not correlate with the proliferative response to gp160, and was not increased by repeated immunization. At the clonal level, the vaccine was shown to elicit a functionally heterogeneous CD4+ T cell response that included clones with antigen-specific, major histocompatibility complex-restricted cytolytic activity. These clones were capable of lysing target cells expressing the HIV-1 env gene and thus might be active against HIV-1-infected cells in vivo.

A recombinant human immunodeficiency virus 1 IIIB (HIV-1IIIB) gp120 subunit vaccine (IIIB-rgp120/HIV-1, Genentech) was tested for safety and immunogenicity in a randomised, double-blind, placebo-controlled phase-I trial. HIV-1-seronegative adult volunteers received three 100 micrograms or 300 micrograms doses of IIIB-rgp120/HIV-1 in alum adjuvant (10 vaccinees in each group), or alum adjuvant alone (8 vaccinees), at 0, 4, and 32 weeks by intramuscular injection. The three injections were well tolerated in both vaccine groups. Antibodies that neutralised homologous HIV-1IIIB were induced in 9 of 10 recipients after three 300 micrograms doses, and 6 of these 9 sera also neutralised heterologous HIV-1SF2. A dose response was evident, since three 100 micrograms injections induced lower titres of HIV-1IIIB neutralising antibodies and in fewer recipients (5 of 9) than the higher dose, with no neutralisation of HIV-1SF2. Similarly, syncytia-inhibiting, CD4-rgp120-blocking, and HIV-1IIIB V3-binding antibodies were induced in a dose dependent manner. Response to the 300 micrograms per dose vaccination occurred in a larger proportion of volunteers and at higher mean titres than seen in previous human trials with other recombinant envelope subunit vaccines or live vaccinia-env priming followed by envelope subunit boosting.

An enzyme immunoassay (EIA) was developed to detect secretory IgA (sIgA) antibodies to HIV-1 envelope glycoprotein, using a mouse monoclonal antibody and a highly purified, baculovirus-expressed recombinant gp160 (rgp160) as antigen. Detection of sIgA was enhanced by prior immunoprecipitation of IgG. IgG and sIgA rgp160 antibodies were measured in parotid saliva and nasal wash samples of 18 HIV-1-seropositive volunteers and 14 HIV-1-seronegative adult volunteers immunized 3 times with HIV-1 IIIB rgp160 vaccine at 1 of 4 dosage levels: 40 micrograms (N = 3), 80 micrograms (N = 3), 160 micrograms (N = 4), and 640 micrograms (N = 4). We detected rgp160-specific IgG antibody in the nasal wash samples of all HIV-1-seropositive volunteers and 4/8 vaccinees (50%) immunized with the two highest doses of rgp160 vaccine. All infected volunteers tested had rgp160-specific sIgA in their nasal wash samples. None of the vaccinees and very few of infected volunteer specimens had detectable antibody in the parotid saliva samples (5/8 had IgG and 1/8 had sIgA). We also detected IgG antibody to rgp160 in the sera of all infected volunteers and 13/14 vaccinees (93%). With this EIA, sIgA antibody can be measured in mucosal secretions of recipients of appropriate candidate HIV-1 vaccines.

A recombinant influenza A vaccine (D protein), comprising a carboxy-terminal sequence from the hemagglutinin HA2 subunit of A/Puerto Rico/8/34 virus (H1N1, A/PR/34) fused to 81 amino-terminal residues of the NS1 nonstructural protein, has previously protected mice against influenza A challenge by inducing H1N1/H2N2 cross-reactive cytotoxic T cells (CTL) without hemagglutination-inhibiting (HI) or neutralizing antibody. In our dose-escalating study, the vaccine was safe in humans and induced both IgG and T cell proliferative responses to D protein but little antibody to A/PR/34 or A/Kawasaki/8/86 (H1N1, A/KW/86) viruses. Among an additional group of A/KW/86-seronegative volunteers immunized with 500 micrograms of D protein, none had a rise in serum HI or neutralizing antibody to A/KW/86, 20% had minimal IgG responses to A/KW/86 by EIA, and a minority had any increase in A/KW/86-specific CTL activity. However, viral shedding and clinical illness score were reduced in vaccines relative to A/KW/86-seronegative unimmunized controls after intranasal challenge with wild-type A/KW/86. D protein immunization conferred significant protective immunity not currently explained by any of the immune parameters measured.

Twelve vaccinia-naive volunteers were inoculated with recombinant vaccinia virus expressing the human immunodeficiency virus type 1 (HIV-1) strain IIIB (HIV-1IIIB) envelope glycoprotein gp160 and subsequently immunized with 640 micrograms of recombinant (r) gp160 protein produced in baculovirus. After booster immunization with rgp160, the sera of all vaccinees showed strong antibody responses detected by Western blot and ELISA; 8 had neutralizing activity and 5 had fusion inhibition activity against the homologous strain; 5 blocked binding of CD4 cells to gp120. Cross-reactive neutralization of HIV-1MN was detected in 3 of 8 sera that neutralized HIV-1IIIB. The combination of live recombinant vaccinia followed by subunit booster immunization was more immunogenic than either product alone and represents a promising approach for HIV-1 immunoprophylaxis. Further definition of recombinant vaccinia safety and augmentation of immune responses to geographically prevalent HIV-1 strains will be necessary before expanding clinical trials to high-risk groups.

The safety and immunogenicity of a human immunodeficiency virus type 1 (HIV-1) gp160 recombinant vaccinia virus (HIVAC-1e) vaccine was evaluated in vaccinia-naive, healthy adults at low risk for acquiring HIV-1 infection. Volunteers (n = 36) were randomized to receive HIVAC-1e or control vaccinia virus at two dosages by bifurcated needle puncture at 0 and 2 months; 12 HIVAC-1e and 6 control vaccinia virus recipients received either 10(6) or 10(7) pfu/mL at each inoculation. There was no significant difference in lesion size, level of viral replication, or systemic symptoms after vaccination with HIVAC-1e or control vaccinia virus. Of 22 HIVAC-1e recipients with lesion formation, 16 developed low-titer gp160-specific antibody responses detectable by Western blot. The peak response occurred between days 70 and 120 and was still detectable at day 365 in 9 of 18 vaccinees. gp160-specific lymphoproliferative responses were detected in 5 of 10 vaccinees. Vaccination with HIVAC-1e was safe in vaccinia-naive, healthy adults and could induce both humoral and cell-mediated gp160-specific immune responses.

The A/Los Angeles/2/87 (H3N2) (A/LA/2/87) virus is sensitive to inhibitors of hemagglutination present in certain human sera. It was found that the effect of these inhibitors could be removed by treating sera with high-concentration receptor-destroying enzyme or trypsin-periodate or by using inhibitor-resistant viruses in the hemagglutination inhibition (HAI) test. Inhibitor-resistant viruses were not effective for detecting rises in antibody titers in the sera of volunteers infected with the A/LA/2/87 wild-type virus, while rises in antibody titer were readily detected in sera treated with trypsin-periodate and tested against A/LA/2/87 wild-type virus in an HAI test. It is therefore suggested that chemical or enzymatic methods be used to inactivate serum inhibitors and that standard virus be used in the HAI test for the currently circulating H3N2 viruses.

Purpose: Histopathology is the standard approach for tissue diagnostics and centerpiece of pathology. Although the current system provides prognostic information, there is need for molecular markers that enhance diagnosis and better predict clinical prognosis. The ability to localize disease-specific molecular changes in biopsy tissue would help improve critical pathology decision making. Direct profiling of proteins from tissue using matrix-assisted laser desorption/ionization imaging mass spectrometry has the potential to supplement morphology with underlying molecular detail. Experimental Design: A discovery set of 11 prostate cancer (PCa)–containing and 10 benign prostate tissue sections was evaluated for protein expression differences. A separate validation set of 54 tissue sections (23 PCa and 31 benign) was used to verify the results. Cryosectioning was done to yield tissue sections analyzed by a pathologist to determine tissue morphology and mirror sections for imaging mass spectrometry. Spectra were acquired and the intensity of signals was plotted as a function of the location within the tissue. Results: An expression profile was found that discriminates between PCa and normal tissue. The overexpression of a single ion at m/z 4,355 was able to discriminate cancer from uninvolved tissue. Tandem mass spectrometry identified this marker as a fragment of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 2 (MEKK2). The ability of MEKK2 to discriminate tumor from normal cells was orthogonally confirmed. Conclusions: This study highlights the potential of this approach to uncover molecular detail that can be correlated with pathology decision making. In addition, the identification of MEKK2 shows the ability to discover proteins of relevance to PCa biology. (Clin Cancer Res 2009;15(17):5541–51)

A cold-adapted (ca) influenza B reassortant vaccine consisting of two genes encoding the hemagglutinin and neuraminidase from wild-type influenza B/Ann Arbor/1/86 virus and the six internal RNA segments from influenza B/Ann Arbor/1/66 ca virus was evaluated in 18 seropositive and 57 seronegative infants and children. The ca reassortant was infectious in seronegative vaccinees, with an estimated 50% human infectious dose of 10(2.5) TCID50. Nasal wash specimens from vaccinees retained the temperature-sensitive phenotype, indicating that the virus was phenotypically stable after replication in fully susceptible children. The vaccine was highly immunogenic in the seronegative vaccinees; 54% of the seropositive vaccinees also developed an increase in serum antibody. The ca vaccine was well tolerated, with only a mild increase in upper respiratory tract symptoms seen in the seronegative vaccinees. These studies indicate that the B/Ann Arbor/1/86 ca reassortant is safe, immunogenic, and phenotypically stable in infants and children.

Cytolytic T lymphocyte (CTL) responses were evaluated in humans immunized with recombinant human immunodeficiency virus type 1 (HIV) envelope glycoprotein gp160. Some vaccinees had gp160-specific CTLs that were shown by cloning to be CD4+. Although induced by exogenous antigen, most gp160-specific CTL clones also recognized gp160 synthesized endogenously in target cells. These clones lysed autologous CD4+ T lymphoblasts infected with HIV. Of particular interest were certain vaccine-induced clones that lysed HIV-infected cells, recognized gp160 from diverse HIV isolates, and did not participate in "innocent bystander" killing of noninfected CD4+ T cells that had bound gp120.

A cold-adapted (ca) influenza B reassortant that derived two genes encoding the hemagglutinin and neuraminidase from influenza B/Ann Arbor/1/86 wild-type virus and six internal RNA segments from ca influenza B/Ann Arbor/1/66 virus was evaluated in 66 adult volunteers having a serum hemagglutination inhibition antibody titer less than or equal to 1:8. The ca reassortant was attenuated and elicited the production of systemic and local antibodies; the 50% human infectious dose was 10(6.4) TCID50. Six weeks after vaccination, 12 unvaccinated volunteers and 13 recipients of ca virus (10(7.5) TCID50) were challenged experimentally with homologous wild-type influenza B virus. The ca vaccine completely protected against illness, and the magnitude of shedding was 50-fold less in vaccinees than in unimmunized controls, five of whom became ill. These findings indicate that the six internal RNA segments of the ca influenza B/Ann Arbor/66 donor virus confer desirable properties of a live virus vaccine to a reassortant derived from a virulent virus. Such reassortants may be suitable vaccines for healthy adults.

Typhoid fever remains an important public health problem in many areas of the world and an effective, non-reactogenic vaccine would be useful to control this disease. An attenuated Salmonella typhi strain (Ty21a), which has shown promise in previous trials, was evaluated in a controlled field trial in Santiago, Chile. In this trial, 82 543 schoolchildren were randomly assigned to receive one or two doses of Ty21a vaccine in enteric-coated capsules or placebo. The enteric-coated vaccine formulation was well tolerated and practical for mass oral immunization. In the first two years of surveillance, 213 cases of bacteriologically-confirmed typhoid fever were found in schoolchildren participating in the trial; annual rates in the placebo group were 139 and 227 per 100 000. Vaccine efficacy in the first two years after vaccination was 59% for two doses and 29% for one dose; no efficacy was found 3–5 years after vaccination. These results indicate that it will be necessary to identify a vaccine formulation and schedule for Ty21a S. typhi that is practical and provides high level protection for > 2 years.

Dabigatran, an oral once-daily unmonitored thrombin inhibitor, has been tested elsewhere using enoxaparin 40 mg once daily. We used the North American enoxaparin 30 mg BID regimen as the comparator. This was a double-blind, centrally randomized trial. Unilateral total knee arthroplasty patients were randomized to receive oral dabigatran etexilate 220 or 150 mg once daily, or enoxaparin 30 mg SC BID after surgery, blinded. Dosing stopped at contrast venography, 12 to 15 days after surgery. Among 1896 patients, dabigatran 220 and 110 mg showed inferior efficacy to enoxaparin (venous thromboembolism rates of 31% [P = .02 vs enoxaparin], 34% [P < .001 vs enoxaparin], and 25%, respectively). Bleeding rates were similar, and no drug-related hepatic illness was recognized. Dabigatran, effective compared to once-daily enoxaparin, showed inferior efficacy to the twice-daily North American enoxaparin regimen, probably because of the latter's more intense and prolonged dosing.

The prostate-specific antigen test has been a major factor in increasing awareness and better patient management of prostate cancer (PCA), but its lack of specificity limits its use in diagnosis and makes for poor early detection of PCA. The objective of our studies is to identify better biomarkers for early detection of PCA using protein profiling technologies that can simultaneously resolve and analyze multiple proteins. Evaluating multiple proteins will be essential to establishing signature proteomic patterns that distinguish cancer from noncancer as well as identify all genetic subtypes of the cancer and their biological activity. In this study, we used a protein biochip surface enhanced laser desorption/ionization mass spectrometry approach coupled with an artificial intelligence learning algorithm to differentiate PCA from noncancer cohorts. Surface enhanced laser desorption/ionization mass spectrometry protein profiles of serum from 167 PCA patients, 77 patients with benign prostate hyperplasia, and 82 age-matched unaffected healthy men were used to train and develop a decision tree classification algorithm that used a nine-protein mass pattern that correctly classified 96% of the samples. A blinded test set, separated from the training set by a stratified random sampling before the analysis, was used to determine the sensitivity and specificity of the classification system. A sensitivity of 83%, a specificity of 97%, and a positive predictive value of 96% for the study population and 91% for the general population were obtained when comparing the PCA versus noncancer (benign prostate hyperplasia/healthy men) groups. This high-throughput proteomic classification system will provide a highly accurate and innovative approach for the early detection/diagnosis of PCA.

The safety, infectivity, and immunogenicity of two human-bovine reassortant rotavirus candidate vaccines were evaluated in adults, children, and infants. One of these, Wa x UK, contained a single human rotavirus gene from the Wa strain that encoded VP4 P1A specificity in a background of 10 bovine genes including the VP7 gene that encodes G6 specificity, whereas the other, Wa x (DS-1 x UK), possessed the human rotavirus VP4 gene from the Wa strain as well as the human VP7 gene from strain DS-1 that encoded G2 specificity. Each of these vaccines appeared to be well-tolerated and immunogenic in infants less than 6 months of age following a single oral dose, and therefore should be evaluated further as vaccine candidates. J. Med. Virol. 66:407-416, 2002. © 2002 Wiley-Liss, Inc.

Resistance to infection with an influenza A virus conferred by previous infection with an influenza A virus belonging to another subtype is called heterosubtypic immunity. Heterosubtypic immunity is demonstrable in laboratory animals but is believed to be weak in humans. The present study examined whether heterosubtypic immunity from previous influenza virus infection induced resistance to infection with an attenuated influenza A vaccine virus. Two groups of vaccinees consisting of young infants and children who received either influenza A H1N1 or H3N2 attenuated virus were studied. Influenza A H3N2 virus vaccine recipients were classified by their preexisting H1N1 heterosubtypic antibody level induced by prior infection with wild-type virus, and the H1N1 vaccinees were classified by their history of infection with H3N2 vaccine virus. For both groups of vaccinees, the rates of seroconversion and virus shedding and the level of vaccine virus replication were compared in subjects with and without heterosubtypic immunity. In 48 influenza A H3N2 virus and 39 H1N1 virus vaccinees, heterosubtypic immunity had no demonstrable effect on infectivity, immunogenicity, or replication of attenuated vaccine virus. These observations confirm the weak nature of heterosubtypic immunity in humans and suggest that it will not limit the utility of live attenuated influenza A viruses in young infants and children.

The safety and immunogenicity of an orally administered, live rotavirus vaccine comprised of four strains, each with a titer of 10(5.3) or 10(5.8) pfu, and each having 10 genes from the UK bovine strain and the VP7 gene from human rotavirus serotype 1, 2, 3, or 4, were evaluated in adults, young children and infants in randomized, double-blind phase 1 trials. Three doses of rotavirus vaccine or placebo given with childhood immunizations to infants at 2, 4, and 6 months of age were well tolerated and did not inhibit antibody responses to childhood vaccines which included DTP, Hib, hepatitis B and OPV. Serum rotavirus antibody responses were detected in 12 of 20 infants after 1 dose, and in 19/19 of the vaccinees after three doses. Neutralizing antibody responses were detected more often against the bovine rotavirus UK strain (95%) than to human rotavirus VP7 serotypes 1 (37%), 2 (32%), 3 (32%) or 4 (32%). The efficacy of this quadrivalent rotavirus vaccine needs to be evaluated further.

A new microparticle enzyme immunoassay (MEIA), the Cytomegalovirus (CMV) Immunoglobulin M (IgM) test, was developed on the Abbott AxSYM analyzer. This test uses recombinant CMV antigens derived from portions of four structural and nonstructural proteins of CMV: pUL32 (pp150), pUL44 (pp52), pUL83 (pp65), and pUL80a (pp38). A total of 1,608 specimens from random volunteer blood donors ( n = 300), pregnant women ( n = 1,118), transplant recipients ( n = 6), and patients with various clinical conditions and disease states ( n = 184) were tested during development and evaluation of this new assay. In a preliminary clinical evaluation we tested specimens collected prospectively from pregnant women ( n = 799) and selected CMV IgM-positive archived specimens from pregnant women ( n = 39). The results from the new CMV IgM immunoassay were compared to the results of a consensus interpretation of the results obtained with three commercial CMV IgM immunoassays. The results for specimens with discordant results were resolved by a CMV IgM immunoblot assay. The relative sensitivity, specificity, and agreement for the AxSYM CMV IgM assay were 94.29, 96.28, and 96.19%, respectively, and the resolved sensitivity, specificity, and agreement were 95.83, 97.47, and 97.37%, respectively. We also tested serial specimens from women who experienced seroconversion or a recent CMV infection during gestation ( n = 17) and potentially cross-reactive specimens negative for CMV IgM antibody by the consensus tests ( n = 184). The AxSYM CMV IgM assay was very sensitive for the detection of CMV IgM during primary CMV infection, as shown by the detection of CMV IgM at the same time as or just prior to the detection of CMV IgG. Specimens from individuals with lupus ( n = 16) or parvovirus B19 infection ( n = 6) or specimens containing hyper IgM ( n = 9), hyper IgG ( n = 8), or rheumatoid factor ( n = 55) did not cross-react with the AxSYM assay. One specimen each from individuals infected with Epstein-Barr virus ( n = 26), measles virus ( n = 10), herpes simplex virus ( n = 12), or varicella-zoster virus ( n = 13) infection, one specimen from an influenza vaccinee ( n = 14), and one specimen containing antinuclear antibody cross-reacted with the assay. The overall rate of cross-reactivity of the specimens with the assay was 3.3% (6 of 184). The AxSYM CMV IgM assay is a sensitive and specific assay for the detection of CMV-specific IgM.

The safety and immunogenicity of the live attenuated cold-passaged, temperature-sensitive (cpts) 248/404 respiratory syncytial virus (RSV) A2 and the RSV A2 purified F glycoprotein (PFP-2) vaccine candidates were evaluated in a placebo-controlled trial in 60 healthy young adults and 60 healthy elderly subjects using simultaneous and sequential (cpts 248/404 followed by PFP-2) vaccination schedules. Both vaccines were well tolerated. The cpts 248/404 vaccine was moderately infectious in both young and old volunteers, but was highly restricted in replication in those who were infected. After both vaccines, RSV neutralizing antibody (neut Ab) titers increased fourfold in 22% of young subjects and in 16% of elderly subjects. Of those with low levels of RSV neut Ab (titer9), 10/12 (83% of) young subjects and six/eight (75% of) elderly subjects had a/=four fold rise in neut Ab titer. Young and elderly subjects immunized simultaneously had similar serum IgG and IgA postimmunization titers to RSV F (IgG, 16.4 vs 16.2, IgA 11.6 vs 12. 5, respectively) as did those who were immunized sequentially (IgG 17.4 vs 17.0, IgA 13.0 vs 13.5). In both age groups, sequential immunization elicited higher postimmunization RSV F IgG and IgA titers than simultaneous immunization. Further studies that combine the PFP-2 subunit vaccine with a less attenuated RSV vaccine should be performed.

The safety and immunogenicity of a novel hepatitis B virus (HBV) vaccine containing recombinant PreS2 and S antigens combined with MF59 adjuvant (HBV/MF59) was evaluated in healthy adults (N=230) who were randomized to receive 2 or 3 immunizations of either the study vaccine or a licensed control vaccine (Recombivax HB®). After a single immunization, 105 of 118 (89%) recipients of HBV/MF59 achieved protective serum levels of anti-HBs antibody (>10 mIU/ml), compared with 13 of 110 (12%) recipients of licensed vaccine (P

Most candidate human immunodeficiency virus (HIV)-1 vaccines induce antibodies that neutralize T cell line-adapted HIV-I strains. Until recently, however, no neutralizing activity against primary HIV-I isolates had been demonstrated in sera from human vaccinees. Since most candidate HIV-I vaccines have been constructed from T cell line-adapted syncytium-inducing (SI) strains, experiments were done to test whether sera from recipients of SI-based vaccines could preferentially neutralize SI primary HIV-1 isolates. Various neutralization assays were performed with sera from volunteers receiving ALVAC gp160MN and/or rgp120 SF2 . Neutralizing activity was detected against 4 of 8 SI primary isolates but against none of 5 non-SI primary isolates. The data suggest that, for the induction of neutralizing antibodies to a broad array of HIV-I primary isolates, a polyvalent vaccine will be needed containing representatives of more than a single category of viruses.

Two live-attenuated, cold-passaged (cp), temperature-sensitive (ts) candidate vaccines, designated cpts530/1009 and cpts248/955, were attenuated, genetically stable, and immunogenic in chimpanzees and were highly attenuated for human adults. In respiratory syncytial virus (RSV)-seropositive children, cpts530/1009 was more restricted in replication than cpts248/955. In seronegative children, 10 4 pfu of cpts248/955 was insufficiently attenuated, and a high titer of vaccine virus was shed (mean peak titer, 10 4.4 pfu/mL), whereas 10 4 pfu of cpts530/1009 was relatively attenuated and restricted in replication (mean peak titer, 10 2.0 pfu/mL). At a dose of 10 5 pfu, cpts530/1009 was immunogenic in seronegative children (geometric mean titer of RSV neutralizing antibodies, 1:724). Transmission of either vaccine to seronegative placebo recipients occurred at a frequency of 20%-25%, Of importance, vaccine viruses recovered from chimpanzees and humans were ts. In contrast to previous studies, this study indicates that live attenuated RSV vaccines that are immunogenic and phenotypically stable can be developed. Additional studies are being conducted to identify a live RSV vaccine that is slightly more attenuated and less transmissible than cpts530/1009.

To determine the immunogenicity of two doses of yeast recombinant hepatitis B virus (HBV) vaccine containing surface (S) protein, an open-label, multicenter trial was conducted in 199 healthy HBV-seronegative adults > or = 40 years old. Volunteers were randomly assigned to 1 of 5 groups to receive a total of three 10-microg doses, at 0, 1, and 6 months, or a total of two doses of 20 microg and 10 microg, 20 microg and 20 microg, 40 microg and 10 microg, or 40 microg and 20 microg at 0 and 6 months. The 40-microg/20-microg regimen elicited the highest rate of seroprotection (96.2%), with a geometric mean titer of antibody against the S protein of 369 mIU/mL, not significantly different from the 536 mIU/mL achieved with three doses. These results suggest that a two-dose regimen can achieve seroprotection similar to that of the three-dose regimen. Whether a shorter interval can be used or a booster dose will be needed later to confer durable immunity are unknown.

Abstract Heterogeneous distribution and function of α 1 -adrenergic receptor subtypes on arterial and venous vessels, together with evidence for altered α-adrenergic receptor expression in hypertension, led us to examine whether mechanical load influences expression of α 1B -and α 1D -adrenergic receptors in rat aortic smooth muscle cells (SMCs). We used RNase protection and radioligand binding assays to measure mRNA and α 1 -adrenergic receptor density. In the first model, SMCs were subjected to phasic loading using flexible culture plates. As a positive control for the load stimulus, postconfluent, quiescent passage 5 cells demonstrated the expected load-dependent morphological realignment. However, no changes were detected in expression of either α 1D - or α 1B -adrenergic receptor mRNAs or receptor density after 24 to 48 hours of loading. β-Actin and SMC-specific α-actin mRNA, as well as cell number and per-cell total RNA and protein, were also unaffected. In a second model, intact thoracic aortas, in either the presence or absence of endothelial cells, were cultured for 48 hours under tonic load. Like cultured cells, 48 hours of load did not affect SMC expression of α 1 -adrenergic receptor mRNAs. We used suprarenal aortic coarctation to examine effects of increased pressure in vivo. As with the previous in vitro and in situ models, hypertension (30 days) had no effect on expression of α 1B - and α 1D -adrenergic receptor mRNAs in the suprarenal aorta compared with sham coarctation. To separate pressure per se from humoral influences, we also measured mRNAs in the subrenal, normotensive aorta. α 1B mRNA levels decreased to 68±14% of sham-coarcted controls in subrenal aorta exposed to normal blood pressure but also to systemic humoral changes induced by coarctation. As a positive control for a load effect, SMC-specific α-actin mRNA increased for loaded aorta in organ culture and in hypertensive aorta in vivo, whereas expression of β-actin mRNA was unaffected. These results from cell culture, organ culture, and in vivo models suggest that pressure (load) alone has no effect on α 1B - and α 1D -adrenergic receptor expression. In coarctation hypertension, smooth muscle protected from the hypertension showed a decline in α 1B mRNA that may be due to a humoral factor or factors.

The inability of antibodies induced by experimental human immunodeficiency virus type 1 (HIV-1) vaccines to neutralize HIV-1 primary isolates may be due to a failure to elicit such antibodies, antigenic differences between the vaccine and the strains tested, insensitivity of the assays used, or to a combination of factors. New neutralization assays were used to determine the ability of candidate AIDS vaccines to generate neutralizing antibodies for clade B primary isolate BZ167, which is closely related in portions of its envelope to the immunizing strains. Sera from HIV-uninfected volunteers in vaccine trials were tested, and neutralizing activity was found in recipients of recombinant (r) gp120 MN or of rgp160 MN -containing canarypox boosted with rgp120 SF-2 . Detection of antibodies that neutralize primary isolate BZ167 correlated with neutralizing activity for homologous vaccine strains. These data demonstrate that certain candidate AIDS vaccines can elicit antibodies that neutralize a primary isolate of HIV-1.

Background. A safe and effective parainfluenza type 3 (PIV-3) virus vaccine is needed to prevent serious PIV-3-associated illness in infants younger than 6 months of age. In previous studies a live bovine PIV-3 (BPIV-3) vaccine, which was developed to prevent human PIV-3 (HPIV-3) disease, was shown to be safe, infectious, immunogenic and phenotypically stable in 6- to 36-month-old infants and children. Methods. The safety, infectivity and immunogenicity of a single dose of the BPIV-3 vaccine was evaluated in a randomized, placebo-controlled, double blinded trial in 19 infants 2 to 5.9 months of age and in 11 additional 6- to 36-month-old subjects. Results. The BPIV-3 vaccine was well-tolerated in both age groups and infected 92% of those younger than 6 months and 89% of those older than 6 months of age. Serum hemagglutination-inhibition (HAI) antibody responses to HPIV-3 and to BPIV-3, respectively, were detected in 42 and 67% of the younger infants, compared with 70 and 85% of the older subjects. In the younger infants we analyzed the rate of antibody response by titer of maternally acquired antibodies; low titer was defined as a preimmunization serum HAI titer

CD8+ cytolytic T lymphocytes (CTL) are likely to be an important component of effective vaccines against human immunodeficiency virus type 1 (HIV-1). CTL can be induced most effectively with live virus vectors. However, because of concerns about the safety of such vectors, a nonreplicating canarypox vector (ALVAC) capable of expressing foreign genes in mammalian cells has been developed. This study evaluated the capacity of an ALVAC vector expressing the HIV-1MN envelope (env) glycoprotein to induce HIV-1-specific CTL in seronegative volunteers. Protocols were designed to determine whether immunization with ALVAC alone or in combination with subunit boosting could induce CTL in vaccinia-immune and -naive volunteers. A simple method for antigen-specific in vitro stimulation was used to detect CTL responses in HIV-1-seronegative vaccine recipients. The results indicate that low doses of a nonreplicating virus vector alone can elicit both CD4+ and CD8+ HIV-1-specific CTL in a subset of seronegative volunteers.