Nasal-associated lymphoid tissue is a site of long-term virus-specific antibody production following respiratory virus infection of mice.

Nasal immunoglobulin A provides an initial defense against inhaled respiratory pathogens. However, it is not known whether the nasal-associated lymphoid tissues (NALT) are able to mount an effective long-lasting pathogen-specific immune response, nor is it known whether functional differences exist between the organized NALT (O-NALT) and the diffuse NALT lining the nasal passages (D-NALT). Here we show that although both the O-NALT and the D-NALT are capable of producing virus-specific antibody in response to influenza virus infection, the frequency of specific antibody-forming cells in the D-NALT is much greater than the frequency observed in the O-NALT. Furthermore, we show that the D-NALT but not the O-NALT is the site of long-term virus-specific humoral immunity which lasts for the life of the animal. These results indicate that the D-NALT is not only the major effector site of the NALT but also the site of local long-term specific antibody production.

Constitutive expression of interleukin 4 in vivo does not lead to the development of T helper 2 type CD8+ T cells secreting interleukin 4 or interleukin 5.

Interleukin 4 (IL-4) has been shown to commit CD8+ T cells to a T helper (Th) 2 functional phenotype in vitro. To study the effects of IL-4 on CD8+ T cell development in vivo we analysed the CD8+ T cell phenotype in mice constitutively expressing IL-4. Purified CD8+ T cells from uninfected or flu infected IL-4 transgenic (tg) animals produced no detectable IL-4 or IL-5 after in vitro stimulation on anti-CD3 coated plates. However, CD8+ T cells from IL-4 tg mice could be converted into IL-4 and IL-5 producers in vitro in the presence of exogenous added IL-4, showing that these cells were still responsive to IL-4. IL-4 tg mice also showed a delay in influenza virus clearance from the lung, which was probably due to the observed reduction of total CD8+ T cell numbers in the IL-4 tg animals since IL-4 tg CD8+ T cells showed normal levels of influenza-specific cytotoxicity in comparison to controls. Taken together these results suggest that CD8+ T cells are not necessarily switched to a Th2 phenotype by the presence of IL-4 and that some other factor(s) may be important in the switch process of CD8+ T cells in vivo, since the addition of IL-4 during CD8+ T cell activation in vitro leads to Th2 type CD8+ T cells secreting IL-4 and IL-5.

ALARA study of teaching effectiveness on reducing radiation exposure.

The purpose of this study was to measure the effectiveness of radiation safety instruction and the impact on radiation film badge levels. A convenience sample of 144 endoscopy nurses and technicians was pretested for radiation safety knowledge, given a course in radiation safety, and then posttested immediately after the course and then 6 months later. Radiation badges were analyzed for radiation exposure at preinstruction, 1 month postinstruction, and 6 months postinstruction. Results showed that the instruction was effective. There was only a slight decrease in radiation badge readings; the decrease, however, was not statistically significant.

Childhood depression, anxiety, and aggression: a reanalysis of Epkins and Meyers (1994).

Research on the constructs of childhood depression and anxiety has typically found that the 2 traits are highly correlated. In a study of the construct validity of childhood depression, anxiety, and aggression, Epkins and Meyers (1994) concluded that, despite some mixed results, the 3 constructs demonstrated adequate convergent and discriminant validity. We reexamined the results of the Epkins and Meyers study in 2 new analyses--a focused comparison of correlational patterns among the traits and a confirmatory factor analysis--and found no support for the discriminant validity of depression and anxiety and only weak support for their convergent validity.

Subverting lymph node trafficking by treatment with the Mel-14 monoclonal antibody to L-selectin does not prevent an effective host response to Sendai virus.

A single 250-micrograms dose of the Mel-14 mAb to L-selectin greatly diminished the extent of L-selectin expression on lymphocytes and decreased (60 to 90%) the massive cellular recruitment to the cervical and mediastinal lymph nodes that follows intranasal infection of naive C57BL/6 mice with Sendai virus. The numbers of CD8+ CTL precursors in the mediastinal lymph nodes were considerably reduced on day 7, when compared with virus-infected mice given a control rat IgG2a, but potent CTL effectors were present in the lungs of both groups by day 10 after infection, and the overall magnitude of CTL precursor generation was not obviously compromised. The early dominance of Sendai virus-specific IgM Ab-forming cells was prolonged in the Mel-14-treated mice, whereas plasma cells producing virus-specific IgA were abnormally prominent in the lymph nodes but not in the spleen. The kinetics of virus-specific Ab-forming cells generation and the serum Ab response for the various IgG isotypes were also delayed. Thus, though L-selectin is clearly important for the localization of naive lymphocytes to regional lymph nodes, the Mel-14-treated mouse can still deal effectively with a virus that causes productive infection only in the respiratory tract. The spleen, where L-selectin does not determine lymphocyte trafficking, is a major site for the compensatory T cell and B cell responses.

Host response to Sendai virus in mice lacking class II major histocompatibility complex glycoproteins.

The development of Sendai virus-specific cytotoxic T-lymphocyte (CTL) effectors and precursors (CTLp) has been compared for mice that are homozygous (-/-) for a disruption of the H-2I-Ab class II major histocompatibility complex glycoprotein and for normal (+/+) controls. The generation of CD8+ CTLp was not diminished in the -/- mice, though they failed to make virus-specific immunoglobulin G class antibodies. While the cellularity of the regional lymph nodes was decreased, the inflammatory process assayed by bronchoalveolar lavage (BAL) of the pneumonic lung was not modified, and potent CTL effectors were present in BAL populations recovered from both groups at day 10 after infection. There was little effect on virus clearance. Production of interleukin-2 by both freshly isolated BAL inflammatory cells and cultured lymph node cells was greatly diminished, though the -/- mice still made substantial levels of gamma interferon. However, treating the mice with a single dose of a monoclonal antibody to this cytokine, at least some of which is made by CD8+ T cells, did not decrease CTLp frequencies. As found previously with CD4-depleted H-2b mice, the development of Sendai virus-specific CD8+ T-cell-mediated immunity is not compromised by the absence of a concurrent class II major histocompatibility complex-restricted response.

Distinctive kinetics of the antibody-forming cell response to Sendai virus infection of mice in different anatomical compartments.

The single-cell ELISPOT assay was used to determine the frequency and isotype commitment of virus-specific antibody-forming cells (AFC) at different anatomical locations following intranasal Sendai virus infection of C57BL/6 and 129/Sv mice. AFC responses in the mediastinal and cervical lymph nodes showed sharp increases and declines, first of IgM AFC, peaking about 7 days after infection, and then of IgG and IgA AFC, peaking about 10 days after infection. A wave of IgM AFC preceding the other isotypes was less evident in the spleen, where peak frequencies of AFC occurred 14 days after infection. Virus-specific AFC appeared in the bone marrow with a unique kinetic pattern, increasing in frequency gradually over the first 3 weeks after infection to a plateau that remained constant. Circulating IgM and IgG achieved significant titers approximately a week after infection; IgM titers were transient, but IgG levels increased sharply and remained high, reflecting the longevity of the bone marrow AFC response. Strain differences in isotype bias were noted, particularly preferential switching to the gamma 2a gene in 129/Sv mice. The B-cell response to acute respiratory viral infection thus exhibits features that are distinct from the primary response to nonreplicating antigens.

Mice lacking CD8+ T cells develop greater numbers of IgA-producing cells in response to a respiratory virus infection.

Antibody production profiles have been compared for Sendai virus infection of normal mice and mice that lack CD8+ T cells as a consequence of treatment with a lymphocyte subset-specific monoclonal antibody or homozygous disruption of the beta 2-microglobulin (beta 2-m(-/-)) gene encoding the light chain of the class I major histocompatibility complex glycoprotein. Using the single-cell ELISPOT assay, we show a relative increase in IgA antibody forming cell (AFC) numbers in the mediastinal lymph node (MLN), spleen, and bone marrow of the CD8-depleted mice. This is reflected in higher serum IgA titers. Similarly, secondary infection with a large dose of Sendai virus leads to greater prevalence of virus-specific IgA AFCs as early as Day 5 postinfection in the beta 2-m(-/-) mice. Also, in primed beta 2-m(-/-) mice challenged with vaccinia constructs containing the genes for the hemagglutinin-neuraminidase (HN), nuclear protein, or the fusion protein of Sendai virus, the majority of the virus-specific AFCs in the MLN are specific for HN and secrete IgA.

Respiratory virus infection of mice provokes a permanent humoral immune response.

We have observed that respiratory virus infection of mice provokes an extremely persistent humoral immune reaction, due to a long-sustained population of antibody-secreting cells in the bone marrow. Theories of humoral immunity that strongly distinguish primary and secondary reactions thus may not adequately describe the immune response to respiratory viruses.

Virus-specific CD8+ T-cell memory determined by clonal burst size.

Although some viruses, particularly the herpes viruses, may never be eliminated from the body, others like influenza A, regularly reinfect humans and boost waning crossreactive CD8+ T-cell immunity. Prolonged T-cell memory is found for viruses that are unlikely to be re-encountered and which do not persist in the host genome, indicating that CD8+ T-cell memory might be independent of continued (or sporadic) antigenic exposure. A feature of virus-specific CD8+ T-cell memory is that antigen-specific cytotoxic T-lymphocyte precursors (CTLp) are greatly increased and remain high throughout life. The idea that persistence of the inducing antigen is essential is based on experiments in which adoptively transferred CD8+ memory T cells could not be detected for more than a few weeks in naive recipient mice without secondary challenge. Here we show that restimulation of such chimaeric mice with an inducing Sendai virus antigen increases the clonal burst size more than 7-fold within 8 days, making memory CTLp easier to detect in the longer term. We find that Sendai-virus-specific CTLp are maintained for > 250 days in irradiated uninfected recipients, including reconstituted beta 2-microglobulin-/- mice. To determine whether a source of viral peptide can persist after primary infection, we gave Sendai-virus-specific Thy1.1+ memory spleen cells to naive mice that had been minimally depleted of Thy1.2+ T cells, or to comparable recipients that had recovered from infection with Sendai virus or influenza virus. Although antibody against Sendai virus was never found in the naive recipients, Sendai-virus-specific CD8+ memory T cells were maintained equally well in each case for > 100 days after cell transfer. We find no evidence for persisting depots of viral protein that might feed into the endogenous processing pathway and maintain virus-specific CD8+ T-cell memory.

Use of high efficacy, lower dose triple therapy to reduce side effects of eradicating Helicobacter pylori.

OBJECTIVES: To evaluate two triple-therapy (TT) regimens of colloidal bismuth subcitrate (CBS), metronidazole, and tetracycline HCl in eradicating Helicobacter pylori, with particular attention to the frequency of resulting adverse effects of the two therapies. METHODS: A prospective, randomized controlled trial was conducted in patients with symptoms of dyspepsia who were positive for H. pylori. Subjects received a 14-day course of either 4 x/day therapy of CBS (108 mg), tetracycline HCl (500 mg), and metronidazole (250 mg), or 5 x/day therapy of CBS (108 mg), tetracycline HCl (250 mg), and metronidazole (200 mg). H. pylori status was determined endoscopically by urease test, histology, and culture. Standard questionnaires were administered to determine compliance to treatment and side effects of therapy. RESULTS: H. pylori was eradicated in 196/213 (92%) patients in the 4 x/day group and 202/210 (96%) in the 5 x/day group (p = 0.07). Side effects were significantly less frequent and less severe in the 5 x/day group (p < 0.01). CONCLUSIONS: We conclude that a lower dose, 5 x/day triple therapy treatment of H. pylori is equally efficacious to the standard 4 x/day therapy, but is accompanied by fewer and milder adverse effects.

Oxygenating mouthguard alleviates hypoxia during gastroscopy.

A randomized study was carried out to determine the effect of oxygen (3 liters/min) via a novel oxygenating mouthguard (Oxyguard) on arterial oxygenation in 242 intravenously sedated patients undergoing gastroscopy. In another group of 21 patients, a randomized crossover study of arterial oxygen saturation using either the standard mouthguard or the oxygenating mouthguard (3 liters/min) was conducted. Significant O2 desaturation (pulse oximeter reading less than 90%) occurred in 25% of patients on room air but only 3% of those on oxygen (p less than 0.001). Severe desaturation (reading less than 85%) occurred in 5% of patients on room air but was prevented by the oxygenating mouthguard. Minimum oxygen saturation levels were significantly higher in patients on oxygen (90.5 +/- 0.3%) than on air (86.5 +/- 0.5%; p less than 0.001). In the crossover group, O2 saturation was uniformly higher in the recordings of all patients using the oxygenating mouthguard. In conclusion, administration of oxygen via the oxygenating mouthguard alleviates hypoxemia during gastroscopy and prevents severe oxygen desaturation. However, hypoxemia may occur even during use of supplemental oxygen. Hence, monitoring of arterial oxygenation is recommended.

Helicobacter pylori-negative duodenal ulcer.

Most patients with chronic duodenal ulcer (DU) craters have gastritis associated with Helicobacter pylori (HP), now thought to be the major cause of DU. A smaller proportion of DU patients have no detectable HP. In this study, we examined the frequency and causes of HP-negative duodenal ulcers. In 302 consecutive patients with endoscopic diagnosis of duodenal ulcer, 284 (94%) were found to have associated HP gastritis, whereas 18 (6%) were HP-negative on histology, culture, and urease test. The largest subgroup of HP-negative patients (8/18) was made up of those who had been taking nonsteroidal antiinflammatory drugs (NSAIDs), followed closely (4/18) by patients with recent intake of antibiotics. Causes of DU in the remaining subgroups included two patients with duodenal Crohn's disease, two with Gastrospirillum hominis infection, one with penetrating carcinoma of the pancreas and one with no detectable cause. We conclude that, although the most common causal factor of duodenal ulcer is HP, some 6% of DU's will be HP-negative, signaling unusual etiology. It is now important to identify the cause of duodenal ulcer so as to initiate appropriate therapy.

Human immunodeficiency virus-1 protease. 1. Initial velocity studies and kinetic characterization of reaction intermediates by 18O isotope exchange.

The peptidolytic reaction of HIV-1 protease has been investigated by using four oligopeptide substrates, Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, Ac-Ser-Gln-Ser-Tyr-Pro-Val-Val-NH2, and Ac-Arg-Lys-Ile-Leu-Phe-Leu-Asp-Gly-NH2, that resemble two cleavage sites found within the naturally occurring polyprotein substrates Pr55gag and Pr160gag-pol. The values for the kinetic parameters V/KEt and V/Et were 0.16-7.5 mM-1 s-1 and 0.24-29 s-1, respectively, at pH 6.0, 0.2 M NaCl, and 37 degrees C. By use of a variety of inorganic salts, it was concluded that the peptidolytic reaction is nonspecifically activated by increasing ionic strength. V/K increased in an apparently parabolic fashion with increasing ionic strength, while V was either increased or decreased slightly. From product inhibition studies, the kinetic mechanism of the protease is either random or ordered uni-bi, depending on the substrate studied. The reverse reaction or a partial reverse reaction (as measured by isotope exchange of the carboxylic product into substrate) was negligible for most of the oligopeptide substrates, but the enzyme catalyzed the formation of Ac-Ser-Gln-Asn-Tyr-Phe-Leu-Asp-Gly-NH2 from the products Ac-Ser-Gln-Asn-Tyr and Phe-Leu-Asp-Gly-NH2. The protease-catalyzed exchange of an atom of 18O from H2 18O into the re-formed substrates occurred at a rate which was 0.01-0.12 times that of the forward peptidolytic reaction. The results of these studies are in accord with the formation of a kinetically competent enzyme-bound amide hydrate intermediate, the collapse of which is the rate-limiting chemical step in the reaction pathway.

Human immunodeficiency virus-1 protease. 2. Use of pH rate studies and solvent kinetic isotope effects to elucidate details of chemical mechanism.

The pH dependence of the peptidolytic reaction of recombinant human immunodeficiency virus type 1 protease has been examined over a pH range of 3-7 for four oligopeptide substrates and two competitive inhibitors. The pK values obtained from the pKis vs pH profiles for the unprotonated and protonated active-site aspartyl groups, Asp-25 and Asp-25', in the monoprotonated enzyme form were 3.1 and 5.2, respectively. Profiles of log V/K vs pH for all four substrates were "bell-shaped" in which the pK values for the unprotonated and protonated aspartyl residues were 3.4-3.7 and 5.5-6.5, respectively. Profiles of log V vs pH for these substrates were "wave-shaped" in which V was shifted to a constant lower value upon protonation of a residue of pK = 4.2-5.2. These results indicate that substrates bind only to a form of HIV-1 protease in which one of the two catalytic aspartyl residues is protonated. Solvent kinetic isotope effects were measured over a pH (D) range of 3-7 for two oligopeptide substrates, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 and Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2. The pH-independent value for DV/K was 1.0 for both substrates, and DV = 1.5-1.7 and 2.2-3.2 at low and high pH (D), respectively. The attentuation of both V and DV at low pH (D) is consistent with a change in rate-limiting step from a chemical one at high pH (D) to one in which a product release step or an enzyme isomerization step becomes partly rate-limiting at low pH (D). Proton inventory data is in accord with the concerted transfer of two protons in the transition state of a rate-limiting chemical step in which the enzyme-bound amide hydrate adduct collapses to form the carboxylic acid and amine products.

Adaptation of the plasma renin radioimmunoassay for use with HIV-1 protease.

We have demonstrated the use of a radioimmunoassay to quantitate the peptidolytic activity of human immunodeficiency virus, type 1 (HIV-1) protease using a tetradecapeptide substrate of porcine renin, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser. HIV-1 protease catalyzes cleavage of this substrate at the same Leu-Leu bond as does porcine renin, resulting in the formation of authentic angiotensin-I. The angiotensin-I product is then detected by use of a commercially available renin plasma assay kit, which constitutes the basis of the RIA. The radioimmunoassay provides detection of the protease-catalyzed formation of angiotensin-I at picomolar concentrations in vitro. We demonstrate the use of this assay in determining IC50 values for two HIV-1 protease inhibitors present in cell culture media and in standard assay buffer. An example of the potential development of this assay for the quantitation of these inhibitors present in ex vivo plasma samples is also presented.

A radiometric assay for HIV-1 protease.

A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.

Inhibition of HIV-1 protease in infected T-lymphocytes by synthetic peptide analogues.

The gag and pol genes of the human immunodeficiency virus type 1 (HIV-1) (ref. 1) are translated as two polyproteins, Pr55gag and Pr160gag-pol (refs 2-6), which are subsequently cleaved by the action of a virus-encoded protease into the four structural gag proteins of the virion core (p17, p24, p7 and p6) and the pol-encoded enzymes essential for retrovirus replication (protease, reverse transcriptase, ribonuclease H, and endonuclease). Mutational inactivation of the proteases of HIV-1 and other retroviruses results in immature, non-infectious virions, indicating that exogenous inhibition of the protease may represent an attractive approach to anti-AIDS therapy. Here we demonstrate that synthetic peptide analogues, which are potent inhibitors of purified HIV-1 protease, inhibit the processing of the viral polyproteins in cultures of HIV-1-infected T lymphocytes and attenuate viral infectivity.

Inhibition of human immunodeficiency virus 1 protease in vitro: rational design of substrate analogue inhibitors.

Inhibitors of the protease from human immunodeficiency virus 1 (HIV-1) were designed, synthesized, and kinetically characterized. Analogues of a heptapeptide substrate of HIV-1 protease with sequence similar to the p17-p24 cleavage site in the natural substrate, Pr55gag, were synthesized in which the scissile dipeptide bond was replaced with bonds from six categories of stable mimics of an aspartic proteolysis transition state or intermediate. These mimics included an analogue of statine, hydroxyethylene isosteres, two categories of phosphinic acids, a reduced amide isostere, and an alpha,alpha-difluoroketone. The resulting peptide analogues were linear competitive inhibitors of purified recombinant HIV-1 protease with inhibition constants ranging from 18 nM to 40 microM depending on the type of inhibitor. A truncated inhibitor, an analogue of a hexapeptide, retained full inhibitory potency. The most potent inhibitors, containing the hydroxyethylene isostere, effectively blocked the proteolytic processing of a recombinant form of Pr55gag by HIV-1 protease in a cell-free assay.

Recurrence of duodenal ulcer and Campylobacter pylori infection after eradication.

The role of Campylobacter pylori gastritis in dyspepsia could be clarified more readily if reliable eradication therapy were available. Antibiotic monotherapy and combined therapy with an antibiotic agent plus a bismuth compound have yielded poor long-term results. In this study, bismuth-tetracycline-metronidazole triple therapy has been used to eradicate C. pylori infection in 100 consecutive patients who were suffering from either a duodenal ulcer or non-ulcer dyspepsia. Examination of a follow-up endoscopic biopsy at eight weeks after treatment showed an eradication rate of C. pylori of 94%. Of 64 patients whose biopsy samples were free of C. pylori infection at eight weeks and who were available for reassessment, 60 (94%) patients had samples that remained free of C. pylori infection on examination of a repeat endoscopic biopsy at 12-37 months (mean, 19.3 months). It is concluded that "triple chemotherapy" can achieve long-term eradication of C. pylori infection effectively in the majority of treated patients and that the recurrence of duodenal ulcers thus may be diminished.