Development and preclinical evaluation of safety and immunogenicity of an oral ETEC vaccine containing inactivated E. coli bacteria overexpressing colonization factors CFA/I, CS3, CS5 and CS6 combined with a hybrid LT/CT B subunit antigen, administered alone and together with dmLT adjuvant.

A first-generation oral inactivated whole-cell enterotoxigenic Escherichia coli (ETEC) vaccine, comprising formalin-killed ETEC bacteria expressing different colonization factor (CF) antigens combined with cholera toxin B subunit (CTB), when tested in phase III studies did not significantly reduce overall (generally mild) ETEC diarrhea in travelers or children although it reduced more severe ETEC diarrhea in travelers by almost 80%. We have now developed a novel more immunogenic ETEC vaccine based on recombinant non-toxigenic E. coli strains engineered to express increased amounts of CF antigens, including CS6 as well as an ETEC-based B subunit protein (LCTBA), and the optional combination with a nontoxic double-mutant heat-labile toxin (LT) molecule (dmLT) as an adjuvant. Two test vaccines were prepared under GMP: (1) A prototype E. coli CFA/I-only formalin-killed whole-cell+LCTBA vaccine, and (2) A "complete" inactivated multivalent ETEC-CF (CFA/I, CS3, CS5 and CS6 antigens) whole-cell+LCTBA vaccine. These vaccines, when given intragastrically alone or together with dmLT in mice, were well tolerated and induced strong intestinal-mucosal IgA antibody responses as well as serum IgG and IgA responses to each of the vaccine CF antigens as well as to LT B subunit (LTB). Both mucosal and serum responses were further enhanced (adjuvanted) when the vaccines were co-administered with dmLT. We conclude that the new multivalent oral ETEC vaccine, both alone and especially in combination with the dmLT adjuvant, shows great promise for further testing in humans.

Establishment of an adult mouse model for direct evaluation of the efficacy of vaccines against Vibrio cholerae.

We describe here a new animal model that offers the prospect of using conventional adult mice for direct evaluation of the protective potential of new cholera vaccines. Pretreatment of adult mice with oral streptomycin allowed intestinal colonization by streptomycin-resistant Vibrio cholerae strains of either the O1 or the O139 serogroup. Bacteria were recovered in greatest numbers from the cecum and large intestine, but recoveries from all regions of the gut correlated significantly with bacterial excretion in fresh fecal pellets, which thus provides a convenient indicator of the extent and duration of gut colonization. Mice immunized mucosally or systemically with viable or inactivated V. cholerae were shown to be comparatively refractory to colonization after challenge with the immunizing strain. Several variables were examined to optimize the model, the most significant being the size of the challenge inoculum; surprisingly, a smaller challenge dose resulted in more consistent and sustained colonization. Studies with mutant strains unable to produce cholera toxin or toxin-coregulated pili revealed that neither factor contributed significantly to colonization potential. Protection against V. cholerae challenge was shown to be serogroup restricted, and significant inverse correlations were detected between serum and intestinal anti-lipopolysaccharide antibody responses and the levels of excretion of challenge organisms.

Various inotropic effects of angiotensin II in post-ischaemic rat hearts depending on ischaemic time with possible involvement of protein kinase C.

AIMS: The present study investigated if the inotropic effect of angiotensin II (AngII) is altered during post-ischaemic reperfusion in hearts subjected to mild and severe ischaemia. The possible involvement of protein kinase C (PKC) in the change in the inotropic effect was also investigated. METHODS: Isolated Langendorff-perfused rat hearts were perfused under constant flow with oxygenated Krebs-Henseleit buffer and paced at 360 beats min(-1). A saline-filled balloon catheter inserted into the left ventricle was used for measurement of contractile force. In the first series of experiments, hearts were subjected to continuous perfusion, 15- or 25-min global ischaemia followed by 45-min reperfusion. At the end of reperfusion, 0.1 micromol L(-1) AngII was infused for 5 min. In a second series of experiments, AngII was infused in hearts subjected to 25-min ischaemia followed by 45-min reperfusion in the absence or presence of the PKC inhibitor chelerythrine chloride (5 micromol L(-1)). RESULTS: The current study demonstrates that AngII exerts a positive inotropic effect in normoxic hearts with an increase of left ventricular developed pressure (LVDP) by 11% (P<0.05 vs. prior to AngII infusion). In post-ischaemic hearts subjected to 15-min ischaemia no effect of AngII was observed. In hearts subjected to 25 min of ischaemia, however, AngII evoked a negative inotropic response with a decrease of LVDP by 18% (P<0.05 vs. prior to AngII infusion). The negative inotropic effect of AngII was inhibited by the PKC inhibitor chelerythrine chloride. CONCLUSIONS: AngII exerts negative inotropic effect in severely injured post-ischaemic heart, possibly through the PKC pathway.

Estimating the size of a transient population.

Migratory populations often stop over for short periods of time at predictable sites along their migration routes. These staging areas can be heavily used and are potentially critical to the survival of the migrants. This paper presents a method for estimating the number of individuals using such an area and their average residence time. The estimator is based on daily population estimates and records of repeat sightings of identifiable individuals. Its application is illustrated with observations on a population of migrating birds, some of which could be identified from bands that were readable from a distance.

Helping clinicians to find data and avoid delays.

One major criticism of paper medical records is the time and effort required to find data items or to gain an overview. Computerisation does not necessarily help. To help clinicians find data faster and with less effort, everyone designing and writing in records needs to understand how and why we search records and the design features that make searching easier. This paper describes how clinicians search medical records and how to improve record design, whether on paper or computer, to help clinicians find all the data they need without delay.

Reading the medical record. II. Design of a human-computer interface for basic reading of computerized medical records.

A user interface for reading the medical record was designed and implemented on a workstation with a 19-inch colour screen. The text is presented on imitations of paper-pages. The pages are organized in bundles which are dynamically connected to scrollable index lists. The turning of pages on the screen is the fundamental concept of the interface. A page can be turned by a mouse-click or by a circular mouse-movement. Elaborated feedback is given to the user in order to provide effortless orientation and navigation. The interface supports the basic ways of use identified in our analyses of reading habits. It also enables human perceptual and cognitive skills to be used. It seems very easy to learn and efficient in use.

Reading the medical record. I. Analysis of physicians' ways of reading the medical record.

Physicians were interviewed about their routines in everyday use of the medical record. From the interviews, we conclude that the medical record is a well functioning working instrument for the experienced physician. Using the medical record as a basis for decision making involves interpretation of format, layout and other textural features of the type-written data. Interpretation of these features provides effective guidance in the process of searching, reading and assessing the relevance of different items of information in the record. It seems that this is a skill which is an integrated part of diagnostic expertise. This skill plays an important role in decision making based on the large amount of information about a patient, which is exhibited to the reader in the medical record. This finding has implications for the design of user interfaces for reading computerized medical records.

Assessment of platelet antibody by flow cytometric and ELISA techniques: a comparison study.

Two different methods for evaluating platelet antibody were used to study 12 normal subjects and 24 patients consisting primarily of intravenous drug users (IVDUs) who were positive for human immunodeficiency virus (HIV). Total platelet-associated immunoglobulin G (IgG) and immunoglobulin M (IgM) were measured by enzyme-lined immunosorbent assay on platelet lysate, and platelet surface-associated IgG and IgM were measured by semiquantitative flow cytometry. IgG and IgM values showed significant correlations between the two measurement methods. Mean platelet surface IgG and total IgG were 3.6 and 4.3 times greater, respectively, in IVDUs than in controls, and platelet IgM was also significantly higher in IVDUs than in controls as measured by both techniques. Although mean platelet immunoglobulin levels were higher in the IVDUs with thrombocytopenia than in IVDUs with normal platelet counts, these differences did not achieve significance. These data show that platelet IgG and IgM levels are increased in IVDU-associated HIV infection and suggest that these increases are not confined to patients manifesting thrombocytopenia. The herein described platelet surface antibody and total platelet antibody measurements appear to be equally useful in studying this patient population. Specific details for generating platelet-associated immunofluorescence units are discussed.

Close relationships between neopterin and beta-2-microglobulin levels in intravenous drug abusers.

Serum from 36 intravenous drug abusers without acquired immunodeficiency syndrome (AIDS) or AIDS-related complex were tested for concentrations of neopterin and beta 2-microglobulin. The seroprevalence of human immunodeficiency virus (HIV) antibody in this group was 50%. Previous studies of this group showed that the HIV antibody positive patients had significant increases in HLA-DR expression on peripheral blood lymphocytes and increases in serum soluble CD8 antigen. Both neopterin and beta 2-microglobulin concentrations were significantly higher in the HIV antibody seropositive patients compared to the seronegative patients (p = 0.001 and p = 0.005, respectively). A highly significant positive correlation between neopterin and beta 2-microglobulin was found for the seropositive patients (r = 0.8879, p less than 0.0001) as well as for the entire group (r = 0.6054, p = 0.0002). Significant positive correlations were also found between neopterin or beta 2-microglobulin and the percent DR + T cells and CD8 antigen levels, although these correlations were not as significant as that observed between neopterin and beta 2-microglobulin. No relationships were found between neopterin or beta 2-microglobulin and total CD4 cell concentrations or CD4/CD8 ratios. These data demonstrate the significant interrelationships between various immune activation markers in a population at risk for developing AIDS.

Levels of soluble CD8 antigen and circulating immune complexes in intravenous drug abusers: relationships to HIV antibody serology.

A total of 36 intravenous drug abusers (IVDA) were studied for circulating immune complexes (CIC) and serum soluble CD8 antigen (sCD8). None had symptoms or signs of AIDS-related complex or AIDS. sCD8 levels were significantly higher in 18 patients who had HIV antibody (Ab) compared with 18 patients who were HIV Ab negative (1640 +/- 578 virus 804 +/- 264 U/ml, p less than 0.0001). In HIV Ab+ patients but not in HIV Ab- patients, sCD8 levels significantly correlated with percentages and absolute numbers of activated CD3+DR+ peripheral blood mononuclear cells (p = 0.0024 and 0.0183, respectively). Also in HIV Ab+ patients, CIC levels were significantly greater for both anti-C3 binding (13.1 +/- 11.1 versus 2.9 +/- 3.4 micrograms/ml, p = 0.002) and C1q binding (23.5 +/- 20.2 versus 6.3 +/- 4.3 micrograms/ml, p = 0.001) CIC. Serum C4 concentrations were lower in the HIV Ab+ patient group (33.9 +/- 10.1 versus 41.6 +/- 12.4 mg/dL, p = 0.043). In the seropositive group, IgG levels were higher (2206 +/- 859 versus 1615 +/- 645 mg/dl) and total CD4 cell counts were lower (757 +/- 344 versus 1172 +/- 402 cells per mm3), but at a less significant level (p = 0.024 and 0.005, respectively), than that seen for sCD8 and C1q CIC differences. These results suggest that elevations of both the lymphocyte activation marker sCD8 and antigen nonspecific CIC characterize earlier stages of HIV infection in IVDA.

T cell immunophenotypes and DR antigen expression in intravenous drug users. Relationship to human immunodeficiency virus serology.

Thirty-six intravenous drug users were studied for peripheral blood mononuclear cell (PBMC) immunophenotypes and human immunodeficiency virus (HIV) serological profiles. This population has a high risk for developing HIV infection. Half (18/36) were HIV antibody (Ab) negative (-) and half were positive (+). Total T lymphocytes (CD3+ and CD2+) were not different between HIV Ab-negative and HIV-positive groups. Unactivated T(CD3+DR-) cells/mm3 were less (p = 0.003) in HIV Ab-positive patients (1,467 +/- 628) compared to HIV Ab-negative patients (2,190 +/- 695). T-helper (CD4+) cells/mm3 were also less in HIV Ab-positive patients (762 +/- 344 vs. 1,161 +/- 419, p = 0.005). The most significant difference was in activated T lymphocyte CD3+DR+) percentages where the mean was 9.6% in those HIV Ab-positive compared to 3.8% in seronegatives (p less than 0.001). Preliminary studies showed that in vitro naloxone treatment of PBMC had no effects on immunophenotypic expression except for CD3+DR+ lymphocytes, where a significant reduction was observed in the HIV Ab-positive group (p = 0.022) but not in the HIV ab-negative group. These findings suggest that in certain populations, activated T cells may be an early manifestation of HIV infection.