Toward Understanding Microbiome-Neuronal Signaling.

Host-associated microbiomes are emerging as important modifiers of brain activity and behavior. Metabolic, immune, and neuronal pathways are proposed to mediate communication across the so-called microbiota-gut-brain axis. However, strong mechanistic evidence, especially for direct signaling between microbes and sensory neurons, is lacking. Here, we discuss microbial regulation of short-chain fatty acids, neurotransmitters, as-yet-uncharacterized biochemicals, and derivatives of neuromodulatory drugs as important areas for assessing microbial interactions with the nervous system.

Cooperative helix stabilization by complex Arg-Glu salt bridges.

Among the interactions that stabilize the native state of proteins, the role of electrostatic interactions has been difficult to quantify precisely. Surface salt bridges or ion pairs between acidic and basic side chains have only a modest stabilizing effect on the stability of helical peptides or proteins: estimates are roughly 0.5 kcal/mol or less. On the other hand, theoretical arguments and the occurrence of salt bridge networks in thermophilic proteins suggest that multiple salt bridges may exert a stronger stabilizing effect. We show here that triads of charged side chains, Arg(+)-Glu(-)-Arg(+) spaced at i,i+4 or i,i+3 intervals in a helical peptide stabilize alpha helix by more than the additive contribution of two single salt bridges. The free energy of the triad is more than 1 kcal/mol in excess of the sum of the individual pairs, measured in low salt concentration (10 mM). The effect of spacing the three groups is severe; placing the charges at i,i+4 or i,i+3 sites has a strong effect on stability relative to single bridges; other combinations are weaker. A conservative calculation suggests that interactions of this kind between salt bridges can account for much of the stabilization of certain thermophilic proteins.

Pharmacokinetics and tolerability of extended-release clarithromycin.

BACKGROUND: Clarithromycin is a semisynthetic macrolide that exhibits broad-spectrum activity against gram-positive, gram-negative, and atypical respiratory tract and skin/skin structure pathogens, Mycobacterium species, and Helicobacter pylori. It is indicated for the treatment of a wide variety of respiratory and dermatologic infections in children and adults as well as prophylaxis and treatment of Mycobacterium avium complex infection and peptic ulcers due to H. pylori. OBJECTIVE: In this article, we review the results of 3 studies of the steady-state pharmacokinetic profiles of clarithromycin and 14(R)-hydroxy-clarithromycin after multiple oral once-daily doses of 500-mg extended-release (ER) clarithromycin tablets. We also review the drug tolerability in 2 phase III comparative clinical trials of immediate-release (IR) and ER clarithromycin conducted in adults with acute maxillary sinusitis (AMS) and acute exacerbation of chronic bronchitis (AECB). METHODS: In the 3 pharmacokinetic studies, multiple-dose regimens of clarithromycin IR (one 250-mg or 500-mg tablet twice daily) and clarithromycin ER (one or two 500-mg tablets once daily), administered to healthy male and female volunteers, were evaluated. The effect of administration in nonfasting versus fasting conditions was assessed as well. Tolerability information was collected from each adult patient enrolled in phase III efficacy studies conducted to support the application for US Food and Drug Administration approval for the treatment of AMS and AECB. Regimens evaluated were 500 mg IR clarithromycin tablets twice daily or 1000 mg (2 x 500 mg) ER clarithromycin tablets once daily for 7 days (AECB) or 14 days (AMS). RESULTS: Bioavailability of the ER clarithromyin tablet administered with food was equivalent to that of the reference IR tablet, based on area under the plasma concentration-time curve (AUC) for both parent compound and active metabolite. The bioavailability of the ER tablet was 30% lower (based on clarithromycin AUC) when administered under fasting versus nonfasting conditions. Compared with the IR tablet, administration of the ER tablet resulted in significantly lower (P < 0.05) clarithromycin peak plasma concentration (Cmax), delayed time to Cmax, and lower degree of concentration fluctuation, confirming its in vivo extended-release characteristics. The most frequently reported adverse events (AEs) in the phase III clinical trials were diarrhea, abnormal taste, and nausea and were generally mild or moderate. The incidence of AEs was comparable for the 2 formulations. The severity of gastrointestinal AEs was significantly less for the ER formulation than for the IR formulation (P = 0.018), as was the frequency of premature study discontinuation due to gastrointestinal AEs or abnormal taste (P = 0.004). CONCLUSIONS: The results from the 3 pharmacokinetic studies reviewed demonstrate the bioequivalence of the ER and IR formulations and support the use of this clarithromycin ER formulation in a once-daily dosing regimen in phase III clinical trials. The ER tablet should be taken with food to maximize bioavailability. The results of 2 phase III comparative clinical efficacy and safety trials of clarithromycin ER tablets versus IR tablets in AMS and AECB confirm the good tolerability of the ER formulation.

Comparison of the efficacy of extended-release clarithromycin tablets and amoxicillin/clavulanate tablets in the treatment of acute exacerbation of chronic bronchitis.

BACKGROUND: Clarithromycin has established efficacy and safety in the treatment of respiratory infections. OBJECTIVE: This study examined the efficacy and safety of a new extended-release formulation of clarithromycin compared with amoxicillin/clavulanate in the treatment of acute exacerbation of chronic bronchitis (AECB). METHODS: This phase IIIB, multicenter, randomized, parallel-group, investigator-blinded study in patients with AECB and productive cough with purulent sputum compared treatment with extended-release clarithromycin (two 500-mg tablets once daily for 7 days) and amoxicillin/clavulanate (one 875-mg tablet twice daily for 10 days). Assessments were performed before treatment, between study days 10 and 12 (or within 48 hours after premature discontinuation), and between study days 17 and 21 (test of cure). RESULTS: Of 287 patients randomized and treated, 270 were clinically evaluable (137 clarithromycin, 133 amoxicillin/clavulanate). Treatment groups were well matched in terms of demographic characteristics, medical condition, and history. Among clinically evaluable patients at test of cure, 85% and 87% of clarithromycin- and amoxicillin/clavulanate-treated patients, respectively, demonstrated clinical cure (as defined in 1998 draft US Food and Drug Administration guidelines); among clinically and bacteriologically evaluable patients, 92% versus 89%, respectively, demonstrated bacteriologic cure. Overall pathogen eradication rates were similar in the 2 groups (88% clarithromycin, 89% amoxicillin/clavulanate). Rates of premature discontinuation of study drug for any reason differed between treatments: 3% (4/142) of clarithromycin-treated patients versus 12% (17/145) of amoxicillin/clavulanate-treated patients (P = 0.005). One percent (2/142) and 6% (8/145) of the respective treatment groups discontinued study drug because of adverse events. Adverse events generally occurred with a similar frequency in the 2 groups; however, taste alteration was more common with clarithromycin (9/142 [6%]) than with amoxicillin/clavulanate (1/145 [1%]; P = 0.01). Mean severity scores for gastrointestinal adverse events showed a significant difference between groups (1.16 for clarithromycin-treated patients and 1.58 for amoxicillin/clavulanate-treated patients: P = 0.016). CONCLUSIONS: The results of this study demonstrate the clinical and bacteriologic equivalence and improved gastrointestinal tolerability of a 7-day course of once-daily extended-release clarithromycin relative to a 10-day course of twice-daily amoxicillin/clavulanate in the treatment of AECB.

Stabilization of alpha-helix structure by polar side-chain interactions: complex salt bridges, cation-pi interactions, and C-H em leader O H-bonds.

It is generally understood that helical proteins are stabilized by a combination of hydrophobic and packing interactions, together with H-bonds and electrostatic interactions. Here we show that polar side-chain interactions on the surface can play an important role in helix formation and stability. We review studies on model helical peptides that reveal the effect of weak interactions between side chains on helix stability, focusing on some nonclassical side-chain-side-chain interactions: complex salt bridges, cation-pi, and C-H em leader O H-bonding interactions. Each of these can be shown to contribute to helix stability, and thus must be included in a comprehensive catalogue of helix stabilizing effects. The issue of the structure of the unfolded states of helical peptides is also discussed, in the light of recent experiments showing that these contain substantial amounts of polyproline II conformation.

Bait ingestion by free-ranging raccoons and nontarget species in an oral rabies vaccine field trial in Florida.

Oral rabies vaccine-laden baits, with a tetracycline biomarker, were distributed in Pinellas County (Florida, USA) by helicopter drop and from cars from January to April 1997. A total of 130,320 baits was distributed throughout the county, yielding an average bait density of 185 baits per km2. Bait ingestion was estimated by microscopic detection of tetracycline in tooth and bone samples from 244 raccoons (Procyon lotor), 33 opossums (Didelphis virginianus), 31 feral cats, and two gray foxes (Urocyon cinereoargenteus) that were trapped during February-April 1997. Active surveillance consisted of 17 trapping sites that were further categorized by six community descriptors. Passive surveillance consisted of animals that were collected as nuisance animals by Pinellas County Animal Services. The proportion of tetracycline positive raccoons was compared between collection techniques, among trapping sites, vegetation communities, and age and sex categories. Since there was no statistically significant difference in the frequency of tetracycline positive raccoons trapped during active surveillance (59%, 110/187) and passive surveillance (53%, 30/57), the data were pooled, resulting in a tetracycline positive frequency of 57% (140/244). The range in the positive tetracycline frequency established for raccoons from the 17 active surveillance sites was 9% (1/11) to 100% (3/3). The tetracycline positive frequency for raccoons ranged from 25% (3/12) at the dumpster sites to 78% (14/18) at the landfills. Juvenile male raccoons (71%, 34/48) were the most commonly marked age and sex class and adult females (42%, 21/50) were the least commonly marked age and sex class. Eighty-five percent (28/33) of the opossums, 3% (1/31) of the feral cats, and 50% (1/2) of the gray foxes were tetracycline positive.

Oral rabies vaccine contact by raccoons and nontarget species in a field trial in Florida.

Rabies is enzootic in raccoons (Procyon lotor) in the eastern United States. Oral vaccination of free-ranging raccoons against rabies has the potential to control the disease in a principal reservoir and reduce the risk of rabies exposure among domestic animals and humans. Free-ranging animal contact with baits containing a vaccinia virus recombinant vaccine expressing the rabies glycoprotein gene (V-RG) was monitored in Pinellas County (Florida, USA) from February through May 1997. Bait contact was assessed with 423 tracking plate nights; conducted in four land use zones: single residential, multiple residential, industrial-commercial, and undeveloped. The undeveloped land use zone was further described by six vegetation communities: mangrove swamp, red maple swamp, beach dune, pine forest, mixed oak hammock, and cabbage palm hammock. Seven animal taxa contacted the baited tracking plates across the four land use zones: raccoons, opossums (Didelphis virginiana), cats (Felis catus), dogs (Canis familiaris), rabbits (Sylvilagus sp.), unidentified rodents, and birds. A total of 252/413 (61%) of the baits was contacted by animals; 95 (38%) of these were specifically by the raccoon, the target species. Overall bait contact by all animals was significantly different among the four land use zones, being highest in the undeveloped zone (82%) and lowest in the industrial-commercial zone (34%). Bait contact by raccoons also was significantly different among the undeveloped and pooled urban zones. Among the six vegetation communities, bait contact by all animals was significantly different ranging from 95% in the mangrove to 50% in the cabbage palm hammock. Among the four vegetation communities tested, bait contact by raccoons also was significantly different.

The effect of antibiotic therapy on bleeding from duodenal ulcer.

OBJECTIVE: We conducted this study to test whether eradication of Helicobacter pylori (H. pylori) infection prevents hemorrhage related to duodenal ulcer. METHODS: Patients with H. pylori infection and endoscopically proven duodenal ulcers without ulcer-related hemorrhage were enrolled into four randomized, double-blind, multicenter studies using the same study protocol. They were treated with clarithromycin plus omeprazole (441 patients), omeprazole alone (447 patients), or ranitidine alone (263 patients). Success of H. pylori eradication was evaluated by the 13C-urea breath test 4-6 wk after the last dose of study drug. Follow-up continued at monthly intervals up to 1 yr after the last dose of study drug. RESULTS: Bleeding due to duodenal ulcer was not observed in any patients who received clarithromycin plus omeprazole, whereas five patients in the omeprazole treatment group and six patients in the ranitidine treatment group experienced an episode of ulcer-related hemorrhage during follow-up. All patients who experienced ulcer-related bleeding were male. When compared by bleeding, there were no significant differences with respect to ethnicity, alcohol consumption, or tobacco use. H. pylori infection was no longer detectable in 68% of patients after treatment with clarithromycin plus omeprazole, compared with 5% after treatment with omeprazole alone or 4% after treatment with ranitidine alone. CONCLUSION: In a population of duodenal ulcer patients without predisposing risk factors for ulcer bleeding, antibiotic eradication or suppression of H. pylori infection prevented the occurrence of ulcer-related hemorrhage for up to 1 yr after therapy.

Effect of H. pylori infection and CagA status on leukocyte counts and liver function tests: extra-gastric manifestations of H. pylori infection.

BACKGROUND: It has been suggested that H. pylori infection is associated with abnormalities in total leukocyte count as well as the number of basophils and lymphocytes. In addition, CagA seropositivity has been associated with an increase in serum transaminase (SGOT) values. The aim of this study was to confirm the findings of previous subgroup analyses in patients before and after treatment for H. pylori infection and to ascertain whether the abnormalities reversed following successful treatment. METHODS: Blood counts and serum transaminase levels were obtained prior to and following treatment of H. pylori infection of H. pylori-infected duodenal ulcer patients. CagA status was assessed by Western blot of the H. pylori isolates obtained from the patients. RESULTS: Ninety-four ulcer patients were studied, including 77 with CagA-positive H. pylori isolates (82%) and 17 with CagA-negative H. pylori isolates. All study parameters remained within normal limits both before and after therapy. There were no significant changes in any study parameter in those who failed therapy. Successful therapy resulted in a significant fall in total white cell count (7413 +/- 520 cmm to 6738 +/- 410 cmm, for pretreatment vs. cured, respectively, p = 0.04) and was almost entirely accounted for by a reduction in the number of circulating polymorphonuclear leukocytes (4595 +/- 370 cmm to 3855 +/- 270 cmm for pretreatment vs. cured, respectively, p = 0.015). The pretreatment SGOT and basophil count were significantly higher in those with CagA-positive H. pylori (SGOT = 23 +/- 1 vs. 18.5 +/- 1 U). Successful or failed therapy with follow-up for 3 months post therapy did not result in a significant change of SGOT levels. CONCLUSIONS: We confirmed an increase in total leukocyte count and number of polymorphonuclear leukocytes in those with H. pylori infection. We also confirmed higher SGOT levels with CagA-positive H. pylori infection, but the failure to resolve within 3 months of cure of the infection makes it unlikely to be a direct result of the H. pylori infection.

Helicobacter pylori does not migrate from the antrum to the corpus in response to omeprazole.

BACKGROUND: Omeprazole is known to have an effect on Helicobacter pylori in vivo. One opinion is that H. pylori "migrates" from the antrum to the corpus in response to omeprazole therapy. METHODS: To determine whether H. pylori migrates in response to omeprazole, we assessed the presence of H. pylori in the antrum and corpus in duodenal ulcer patients receiving omeprazole for 4 wk. Culture and histological examination of antral biopsies (Genta stain) were performed before patients received omeprazole, at the end of therapy, and 4-6 wk later. The end points were presence or absence of H. pylori and the number of H. pylori colonies per biopsy. RESULTS: Seventy-two patients had H. pylori in both the antrum and corpus at entry and 4-6 wk after ending therapy. Three general patterns were prevalent at the end of omeprazole therapy: antrum- and corpus-positive (54%), antrum-negative and corpus-positive (24%), both antrum- and corpus-negative (21%), and one patient had antrum-positive with corpus-negative (1%). Evaluation of the number of colonies per biopsy in those who remained H. pylori-positive in both the antrum and corpus throughout showed that the number of H. pylori decreased in both the antrum and corpus during therapy (507 +/- 60 vs. 225 +/- 51, p < 0.01 and 415 +/- 58 vs. 290 +/- 46 0.1) for antrum and corpus, respectively, and tended to return to pre-therapy levels 4-6 wk later. The number of H. pylori in the corpus also decreased in the antrum-negative and corpus-positive group during therapy with omeprazole (433 +/- 87 vs. 185 +/- 61, p < 0.05). In most of the patients studied, the number of H. pylori in the corpus was less posttreatment than it was pretreatment. The decrease in H. pylori load was also reflected in the development of false-negative urea breath tests. CONCLUSIONS: Omeprazole is detrimental to H. pylori in both the antrum and the corpus; migration from the antrum to the corpus in response to omeprazole is a myth.

Quantitation of cytomegalovirus DNA and characterization of viral gene expression in bronchoalveolar cells of infected patients with and without pneumonitis.

Cytomegalovirus (CMV) is often present in bronchoalveolar lavage (BAL) fluid of immunosuppressed patients without CMV pneumonitis. The amount of viral DNA within BAL cells of patients with definite CMV pneumonitis and of viral shedders was quantitated by polymerase chain reaction (PCR) and the extent of CMV gene expression within BAL cells was defined by reverse transcription - PCR. No viral DNA was detected in 6 viral shedders, and 12 had low copy numbers (mean, 72 copies/10(5) BAL cells; median, 20) compared with numbers in pneumonitis patients (267,580 and 57,000, respectively). When CMV intranuclear inclusions were absent within BAL cells of patients with pneumonitis, copy numbers (mean, 9362; median, 7110) were still significantly higher than among shedders. Expression of viral glycoprotein H mRNA was detected in BAL cells of all 11 pneumonitis patients tested but in 0 of 18 viral shedders. Thus, high-grade infection and viral replication within BAL cells are integral features of CMV pneumonitis but not viral shedding.

Quantitation of human cytomegalovirus glycoprotein H gene in cells using competitive PCR and a rapid fluorescence-based detection system.

A technique is described for quantitation of the human cytomegalovirus (HCMV) glycoprotein H (gH) gene in cells using a quantitative-competitive polymerase chain reaction (QC-PCR). Two recombinant DNA molecules, differing in size due to a 92-bp deletion within the HCMV gH sequence, were used in co-amplification studies to construct a standard curve from which the copy number of the gH gene present in clinical samples could be interpolated. The use of primers labeled with a fluorescent dye allowed direct detection of the amplified products by measuring the amount of fluorescence emitted by each specific PCR fragment with an automated DNA sequencer coupled to a software program. This system was validated subsequently using bronchoalveolar lavage cells obtained from immunocompromised patients and found to be highly sensitive and reproducible over a range of 5-50,000 HCMV gH copies. This rapid procedure could easily be applied to study the pathogenesis of HCMV infection, identify the patients at high risk of developing HCMV disease, and monitor the effects of antiviral therapy at the molecular level.

Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells.

The bone morphogenetic proteins (BMPs) are a group of transforming growth factor beta (TGF-beta)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57- to 62-kDa) and type II (75- to 82-kDa) receptor components for TGF-beta and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs.

Limited T cell response to donor MHC peptides during allograft rejection. Implications for selective immune therapy in transplantation.

Previously, we have demonstrated that during allograft rejection, MHC molecules of the donor are processed and presented to alloreactive CD4+ T lymphocytes in the form of peptides associated with the MHC class II molecules of the recipient. There is an increasing body of evidence that this indirect pathway of allorecognition may play a major role in allograft rejection. Herein, we have used a series of overlapping MHC peptides progressing along the sequence of the donor MHC molecule in single residue steps. We have mapped all potential MHC Ag determinants to which T cell responses could be generated after s.c. injection of allogeneic splenocytes. We have shown that splenic T cell proliferative responses to the beta 1 domains of donor Ak, Ad, and A(s) mouse MHC class II molecules were directed toward a single immunodominant determinant in each of three donor/recipient combinations. Interestingly, after allogeneic spleen cell transplantation, an additional determinant on donor MHC could be detected in the draining lymph nodes. This result shows that the fine specificity of T cell response to donor transplantation Ags can differ between lymphoid organs. Then, we investigated whether limitation of T cell response to donor MHC peptides applies to the clinical situation of a graft. We have shown that after an allogeneic skin graft, self-restricted alloreactive T cells proliferated to the same determinant on the donor MHC molecule. These results indicate that immune intervention, such as tolerance induction to the dominant T cell determinant on donor MHC molecules, may be developed for the prevention of allograft rejection.

Disruption of the determinant hierarchy on a self-MHC peptide: concomitant tolerance induction to the dominant determinant and priming to the cryptic self-determinant.

The presentation of self-peptides in a self-restricted manner plays a critical role in the complex positive and negative selective process of T cell recognition of self-determinants. The population of determinants comprises (i) a dominant set which is efficiently presented and induces clonal elimination or inactivation of the corresponding autoreactive T cells, and (ii) a cryptic set which is not processed efficiently enough to reach the threshold of presentation to make an impact on the T cell repertoire during thymic selection. Here we have studied a self-MHC peptide, Ld 61-85, which as shown in earlier work was able to induce vigorous T cell proliferation in syngeneic animals. Despite the fact that this peptide as a whole is 'cryptic', the fine specificity of the class II restricted response was complex, in that there were three distinct and overlapping T cell determinants: the dominant determinant, Ld 65-80, flanked by two cryptic determinants, Ld 61-75 and Ld 73-85, all of which compete for stimulating in vivo autoreactive T cell proliferative responses. The hierarchy of these determinants bears an interesting relationship to tolerance. Ld 61-85 or Ld 61-80 priming induces proliferation only to Ld 65-80; likewise, tolerance induction to Ld 61-80 prevents elicitation of a subsequent response to Ld 61-80 or Ld 65-80 in the local lymph nodes. However, in the Ld 61-80 tolerant mice, in vivo challenge with Ld 61-80 induces a strong T cell proliferative response directed towards cryptic Ld 61-75.(ABSTRACT TRUNCATED AT 250 WORDS)