Transcriptome analysis of severely active chronic spontaneous urticaria shows an overall immunological skin involvement.

BACKGROUND: The knowledge about chronic spontaneous urticaria (CSU) phenotypes is based on its clinical characteristics, associated comorbidities, course of the disease, and its response to the available effective drugs. Genotype expression and its further correlation with CSU phenotypes are still unknown. We describe the cutaneous transcriptome of patients suffering a severely active CSU refractory to antihistamine treatment. METHODS: Through the bioinformatic analysis of the whole Human Genome with Oligo Microarrays and quantitative real-time polymerase chain reaction (qPCR), relevant genes expressed in nonlesional (NLS-CSU) and lesional skin (LS-CSU) and peripheral blood were identified in 20 patients suffering from severely active CSU and 10 healthy controls (HCs). RESULTS: From 39 genes differentially expressed in NLS-CSU when compared with HCs, 31 (79.48%) were confirmed by qPCR corresponding to genes involved in epidermal homeostasis and dermal repair. From the analysis comparing LS-CSU with NLS-CSU, a selection of 142 genes was studied with qPCR, and 103 (72.53%) were confirmed. Differentially expressed genes in the phenomenon of wheal development are involved in a variety of biological functions as, epidermal differentiation, intracellular signal function, transcriptional factors cell cycle differentiation, inflammation, or coagulation. Differentially expressed genes that uniformly increase or decrease along the skin worsening until the wheal appearance is shown. CONCLUSION: The skin of CSU patients with a severely active disease shows an overall immunological skin involvement showing a peculiar gene profile.

[Recurrence of Clostridium difficile-associated diarrhoea prevented by the administration of a whey concentrate from specifically immunised cows; prospective study]. / Recidieven van Clostridium difficile-geassocieerde diarree voorkómen door toediening van een weiconcentraat van specifiek geïmmuniseerde koeien; prospectief onderzoek.

OBJECTIVE: To try to prevent recurrences of Clostridium difficile-associated diarrhoea (CDAD) by treatment with a specific neutralising secretory IgA-enriched whey-protein concentrate (40%) made from the milk of cows immunised with C. difficile and its toxins. DESIGN: Prospective, non-blinded, clinical cohort study. METHOD: In 2005-2006, 100 consecutive patients with CDAD received the whey concentrate for 2 weeks after completion of standard antibiotic therapy. For a period of 60 days after the start of the administration, the safety and preliminary efficacy of the whey concentrate were evaluated by means of a diary, blood determinations, active surveillance for adverse events, and the recurrence of CDAD. RESULTS: The whey concentrate was well tolerated and no safety issues were raised. Eleven out of 109 episodes (10%) were followed by a recurrence. After completion of the whey concentrate therapy, a positive test for faecal toxins or culture of C. difficile was predictive for the recurrence of CDAD (relative risk: 8.2 (95% CI: 1.04-64), and 4.7 (95% CI: 0.5-47), respectively). A positive faeces toxin during administration of the whey concentrate was also associated with an early recurrence of CDAD. CONCLUSION: Compared to historical and contemporary findings in control groups, the whey concentrate appeared to reduce the recurrence of CDAD by about 50%. However, the standard dose of the whey concentrate was probably not sufficient to fully neutralise the C. difficile toxins in faeces in all episodes.

Community-onset Clostridium difficile-associated diarrhoea not associated with antibiotic usage--two case reports with review of the changing epidemiology of Clostridium difficile-associated diarrhoea.

The emergence of hypervirulent strains of Clostridium difficile causing outbreaks in hospitals and nursing homes may result in a greater than before spread of the bacterium in the community. By consequence, the incidence of community-onset cases of Clostridium difficile-associated diarrhoea (CDAD) may increase outside known risk groups that are currently characterised by prior hospitalisation, prior antibiotic usage, older age and significant comorbidity. Here, we describe two case histories of community-onset CDAD. The first concerns a previously healthy young female with community-acquired CDAD without recent hospitalisation or antibiotic usage. The second patient developed diarrhoea in the community after discharge from a hospital where--in retrospect--an outbreak of CDAD occurred. The cases illustrate that CDAD should be included in the differential diagnosis of patients seeking care for community-onset diarrhoea, even in those without characteristic risk factors for CDAD.

Wide-range transcriptional modulating effect of ntrR under microaerobiosis in Sinorhizobium meliloti.

A mutation in the second gene in the ntrPR operon results in increased expression of nodulation (nod) and nitrogen fixation (nif) genes in Sinorhizobium meliloti. Since this pleiotropic effect is particularly pronounced in the presence of external combined nitrogen, a nitrogen regulatory function has been suggested for NtrR. To identify the complete set of protein-coding genes influenced by loss of ntrR function, microarray hybridizations were carried out to compare transcript levels in the wild type and mutant strains grown under aerobic and microaerobic conditions. Of the 6207 genes examined, representing the entire genome of S. meliloti, 7% exhibited altered expression: 4.5% of the genes are affected under oxic, 2.5% under microoxic conditions. 0.4% of all the genes are affected under both oxygen concentrations. A microoxic environment is required for the induction of genes related to symbiotic functions but results in the down-regulation of other (e.g. metabolic) functions. When the alterations in transcription levels at low oxygen concentration in the mutant strain were compared to those of the wild type, a modulating effect of the ntrR mutation was observed. For example, symbiotic nif/fix genes were induced in both strains, but the level of induction was higher in the ntrR mutant. In contrast, genes related to transcription/translation functions were down-regulated in both strains, and the effect was greater in the wild-type strain than in the ntrR mutant. A relatively wide range of functions was affected by this modulating influence, suggesting that ntrR is not a nitrogen regulatory gene. Since genes encoding various unrelated functions were affected, we propose that NtrR may either interfere with general regulatory mechanisms, such as phosphorylation/dephosphorylation, or may influence RNA stability.

Paradigm shifts in clinical trials enabled by information technology.

The use of the world wide web for clinical trials changes the processes of performing clinical research in several fundamental ways. Greatly improved security, monitoring capability, and accuracy and timeliness of study conduct can be achieved while lowering cost. Data quality is enhanced while co-ordinating centre effort is reduced. The web provides a natural environment for linking the various components of clinical research, leading to new levels of simplicity and efficiency. It also enhances opportunities for recruitment of study investigators and patients. Other information technology tools and databases can be used to assist in this regard as well. Web-based trials change the relationship of the investigator site to the study and the site to the co-ordinating centre. Different roles and responsibilities lead to simplified processes and more and higher quality data. Many standard co-ordinating centre activities, such as randomization, protocol implementation and amending, document tracking, adverse event reporting, site monitoring, report generation and data analysis are all fundamentally changed in a web-based trial. Opportunities are enhanced to identify potential investigators and support their successful study conduct. As the role of investigator sites is changed in web-based research, more primary care medical providers can be attracted to research, providing more typical patients to studies than those sometimes available through more traditional research sites, especially those at academic study sites. Other activities can now be co-ordinated electronically with the advent of the web. The Institutional Review Board (IRB) can use online tools to control investigator participation, resulting in improved study efficiency and patient safety. A web-based research pharmacy provides tremendous efficiencies in managing and distributing study medications. Financial payments to the sites can be performed and recorded electronically, or even administered based on timeliness and quality of the data. Our early experience with web-based trials indicates that there can be tremendous gains in study efficiency and accuracy by restructuring processes, roles and responsibilities through a comprehensive centralized, web-based trial. The future appears bright for web-based clinical trials.

Detecting dose response with contrasts.

Analyses of dose response studies should separate the question of the existence of a dose response relationship from questions of functional form and finding the optimal dose. A well-chosen contrast among the estimated effects of the studied doses can make a powerful test for detecting the existence of a dose response relationship. A contrast-based test attains its greatest power when the pattern of the coefficients has the same shape as the true dose response relationship. However, it loses power when the contrast shape and the true dose response shape are not similar. Thus, a primary test based on a single contrast is often risky. Two (or more) appropriately chosen contrasts can assure sufficient power to justify the cost of a multiplicity adjustment. An example shows the success of a two-contrast procedure in detecting dose response, which had frustrated several standard procedures.

Providing evidence of efficacy for a new drug.

There are many issues to consider when designing an efficacy package for drug registration. Generally in Europe and the United States, two or more confirmatory trials demonstrating efficacy (p < 0.025, one-tailed) of the test treatment versus a suitable control group must be conducted with a priori definition of a primary efficacy endpoint. Exceptions are possible, and there is always extensive discussion whenever less is proposed or more is required. Every aspect of the basic requirement can be questioned: number of trials; choice of control groups; selection of primary efficacy variables(s); levels of significance; one-tailed versus two-tailed test. These issues will be discussed, and justification is given when proposals are made for deviations from standard practice. Differences between Europe and the U.S. are discussed for certain disease entities. Because the assessment of the weight of evidence in favour of a drug effect is difficult to quantitate, if not impossible, no definitive guidance can be given that is suitable for all circumstances and countries.

The regulatory protein MucR binds to a short DNA region located upstream of the mucR coding region in Rhizobium meliloti.

The Rhizobium meliloti MucR protein is known to regulate the biosynthesis of the two exopolysaccharides, succinoglycan and galactoglucan. The mucR gene was successfully overexpressed in Escherichia coli BL21 cells by heat shock induction using a two-plasmid system. Cell extracts of the production strain contained about 20% of a polypeptide of 17 kDa apparent molecular mass, corresponding to the size expected for MucR. As shown by an electrophoretic mobility shift assay, these extracts were active in the specific retardation of a 219-bp DNA fragment including 134-bp of the non-coding region upstream of the mucR gene. Primer extension analysis showed that this DNA fragment was located within the transcribed region upstream of the mucR gene. Competition experiments revealed that a 44-bp sequence present within the 134-bp upstream of the mucR gene contained the MucR binding site. Although binding of MucR to this site exhibited a moderate dissociation constant of Kd approximately 1.4 x 10(-7) M, the reaction was highly specific since fragments containing binding sites for the homologous Ros protein from Agrobacterium tumefaciens were not able to compete for MucR binding.

The 32-kilobase exp gene cluster of Rhizobium meliloti directing the biosynthesis of galactoglucan: genetic organization and properties of the encoded gene products.

Proteins directing the biosynthesis of galactoglucan (exopolysaccharide II) in Rhizobium meliloti Rm2011 are encoded by the exp genes. Sequence analysis of a 32-kb DNA fragment of megaplasmid 2 containing the exp gene cluster identified previously (J. Glazebrook and G. C. Walker, Cell 56:661-672, 1989) revealed the presence of 25 open reading frames. Homologies of the deduced exp gene products to proteins of known function suggested that the exp genes encoded four proteins involved in the biosynthesis of dTDP-glucose and dTDP-rhamnose, six glycosyltransferases, an ABC transporter complex homologous to the subfamily of peptide and protein export complexes, and a protein homologous to Rhizobium NodO proteins. In addition, homologies of three Exp proteins to transcriptional regulators, methyltransferases, and periplasmic binding proteins were found. The positions of 26 Tn5 insertions in the exp gene cluster were determined, thus allowing the previously described genetic map to be correlated with the sequence. Operon analysis revealed that the exp gene cluster consists of five complementation groups. In comparison to the wild-type background, all exp complementation groups were transcribed at a substantially elevated level in the regulatory mucR mutant.

Randomization tests for assessing the equality of area under curves for studies using destructive sampling.

Testing the equality of the area under a curve (AUC) for different dose groups is frequently done in pharmacokinetic research. Equality of AUCs is one indicator of bioequivalence. When the experimental unit must be sacrificed to obtain a response, AUC can be simply estimated using a linear combination of response means at various time points. The distribution of this estimator is simply obtained using standard statistical theory, and statistical hypothesis tests are easily constructed. These tests assume a normal distribution of responses at each time point (or at least large enough samples to assure that the mean response is normally distributed). The applicability of this test to cases of non-normal response distributions when small numbers of observations are sampled at each time point is questionable. Randomization tests are suggested for this problem. These tests provide a valuable alternative to this normal-theory test. Discussion of the assessment of dose proportionality is also presented.

Factorial dose-response studies using frequency and magnitude of dose.

In the early stages of traditional drug development, the frequency of dosing (e.g., QD, BID, etc.) is typically determined by the pharmacokinetic properties of a compound. After an appropriate dose frequency is chosen, the magnitude of dose is then evaluated via parallel-group dose-response trials. For some drugs, however, blood levels at any given time may not be accurate predictors of clinical response, or the drug may not be absorbed systemically. In those instances, we propose the use of a factorial dose-response trial that simultaneously evaluates frequency of dosing and magnitude of dose. We consider this approach to selecting an appropriate dosing regimen to be more scientifically founded and more cost-effective, than independent evaluation of dose and frequency through separate clinical trials. Some design considerations and statistical analysis strategies for these factorial trials are presented in this paper.

Dose-response relation between terfenadine (Seldane) and the QTc interval on the scalar electrocardiogram: distinguishing a drug effect from spontaneous variability.

The primary goal of this investigation was to describe the effect of terfenadine on the QT interval corrected for heart rate (QTc) of the scalar electrocardiogram (ECG). The design was double-blind, four-period crossover, dose escalation, which involved 28 normal healthy volunteers and 28 patients with stable cardiovascular disease. At baseline, the normal subjects had a mean QTc interval of 407 msec, whereas the patients with cardiovascular disease had a mean QTc interval of 417 msec (p<0.01). The largest increase in mean QTc on terfenadine was 24 msec in a normal subject and 28 msec in a patient with cardiovascular disease. The longest average QTc observed was 449 msec and 501 msec in any normal subject and patient with cardiovascular disease, respectively. Compared to baseline, terfenadine 60 mg twice daily is associated with a QTc increase of 6 msec in normal subjects and a 12 msec increase in patients with cardiovascular disease (p<0.01 vs baseline; p>0.05 when the two populations were compared). Although the QTc increase from baseline are statistically significant, the magnitude of the spontaneous variability in QTc in the same patients is much greater. Because 40 ECGs were obtained while taking placebo in each participant, the spontaneous variability in QTc interval with placebo was also described. Only one of the 28 normal subjects had a mean baseline QTc=440 msec, yet 14 of the 28 normal subjects had at lease one of the 40 placebo ECGs with a QTc=440 msec. The 28 patients with cardiovascular disease had a mean QTc at baseline of 417 msec; yet 20 of 28 had at lease one ECG on placebo with a QTc interval = 440 msec. On the average, the QTc fluctuated 56 msec in each patient during placebo administration. From the observed placebo variability, we calculated that an increase in QTc of=35 msec while receiving drug therapy is likely to represent a drug effect at the 95% confidence interval.

A homolog of the Rhizobium meliloti nitrogen fixation gene fixN is involved in the production of a microaerobically induced oxidase activity in the phytopathogenic bacterium Agrobacterium tumefaciens.

Hybridization analysis using the Rhizobium meliloti nitrogen fixation gene fixN as a probe revealed the presence of a homologous DNA region in the phytopathogenic bacterium Agrobacterium tumefaciens. Hybridization signals were also detected with total DNAs of Rhizobium leguminosarum bv. phaseoli, Rhodobacter capsulatus and Escherichia coli, but not those of Xanthomonas campestris pv. campestris and Pseudomonas putida. The hybridizing fragment from A. tumefaciens was cloned and sequenced. The predicted gene product of one of the two open reading frames identified on the sequenced fragment shows homology to FixN of different Rhizobiaceae as well as a low but significant similarity to subunit I of heme copper oxidases from various bacteria. The presence of five strictly conserved histidine residues previously implicated in forming ligands to heme and CuB in oxidases and the predicted membrane topology provide evidence that the A. tumefaciens fixN-like gene product is a component of the heme copper oxidase superfamily. The incomplete open reading frame starting only 8 nucleotides downstream of the fixN-like gene exhibits homology to Rhizobium fixO. Using an uidA (GUS) gene fusion it could be shown that the A. tumefaciens fixN-like gene is preferentially expressed under microaerobic conditions. Expression of the uidA fusion is abolished in R. meliloti fixJ and fixK mutants, indicating that an Fnr-like protein is involved in transcriptional regulation of the fixN-like gene in A. tumefaciens. The presence of an upstream DNA sequence motif identical to the Fnr-consensus binding site (anaerobox) further supports this hypothesis. A. tumefaciens mutated in the fixN-like gene shows decreased TMPD-specific oxidase activity under microaerobic conditions, indicating that the fixN-like gene or operon codes for proteins involved in respiration under reduced oxygen availability.

Dose response studies. I. Some design considerations.

A critical aspect of biomedical research is the characterization of the dose response relationship of a compound. This is true in laboratory experiments and clinical trials and pertains to efficacy, safety, and the resulting benefit/risk ratio. Presented here is Part I of this article, which deals with some clinical trial design issues surrounding dose response studies. Some additional comments are made about trials for identifying the minimum effective dose, randomized concentration controlled trials, and the use of one-sided hypotheses in designing such trials. Part II is a separate paper reviewing some analysis strategies for dose response studies.

Dose response studies. II. Analysis and interpretation.

The primary focus of this paper is to examine analysis strategies for parallel, randomized dose response studies with particular emphasis on identifying the minimum effective dose. Such studies have become a standard for drug development in the pharmaceutical industry. Particular attention is paid to ANOVA followed by multiple comparison procedures with some additional discussion of the utility or regression models. When there are three or fewer dose groups and a placebo in a study, ANOVA techniques are preferred; with a larger number of dose groups, regression analysis has greater utility and reliability. Analysis of factorial dose response studies is reviewed only slightly as this is an emerging area of interest, and further development is necessary.

Applicability of teicoplanin dosage adjustment guidelines for renally impaired patients over the range of 3 to 30 mg kg-1.

The pharmacokinetics of teicoplanin were investigated in 13 subjects with various degrees of renal impairment using a randomized two-period crossover design; 11 subjects completed both periods. Doses of 3 and 30 mg kg-1 were administered as single dose, 60-min constant rate intravenous infusions. Blood samples were obtained over 28 days and urine was collected over 48 h. Serum and urine were analyzed using a microbiological assay. As previously observed in studies conducted in renally impaired subjects, teicoplanin total and renal clearance significantly decreased with decreasing creatinine clearance (p < 0.0001). However, for these parameters, no differences between doses were observed. Dosage adjustment guidelines for renally impaired patients are usually developed using the ratio of total clearance in renally impaired patients to the total clearance in patients with normal renal function. Since no dose-related differences existed in the relationship between teicoplanin total clearance and creatinine clearance, initial dosage adjustment guidelines for renally impaired patients developed at 3 or 30 mg kg-1 are applicable over the range of 3 to 30 mg kg-1.

Pharmacokinetics of terfenadine in healthy elderly subjects.

The pharmacokinetics of the terfenadine active metabolite, metabolite I, was examined in ten healthy elderly adults and ten younger adults after single-dose oral administration of 120-mg terfenadine. All subjects successfully completed the study without reporting sedation or other adverse events. Absorption was rapid in both the young and elderly. The mean Cmax was the same for both groups, 501 ng/mL, and occurred at 2.3 hours in the young subjects and 2.5 hours in elderly subjects. However, the apparent clearance was reduced by about 25% in the elderly. After correcting clearance for bodyweight, this difference was not statistically significant.

Pharmacokinetics of teicoplanin upon multiple-dose intravenous administration of 3, 12, and 30 milligrams per kilogram of body weight to healthy male volunteers.

Teicoplanin pharmacokinetics were evaluated after multiple-dose intravenous administration to healthy male volunteers by using a randomized, double-blind, parallel design. Doses of 3, 12, or 30 mg of teicoplanin per kg of body weight were administered every 24 h for 14 days as 60-min constant-rate intravenous infusions. Blood and urine samples were collected over 21 days and analyzed by a microbiological assay. Twenty-three subjects were included in the pharmacokinetic analysis. The median pharmacokinetic parameters upon multiple-dose intravenous administration of 3, 12, and 30 mg/kg included steady-state volumes of distribution of 0.94, 0.77, and 0.68 liter/kg; total clearances of 11.9, 12.0, and 13.2 ml/h/kg; and terminal disposition half-lives of 143, 166, and 96 h, respectively. Renal clearance accounted for approximately 95% of total clearance. No dose-related differences existed for teicoplanin total or renal clearance. The steady-state volume of distribution decreased significantly with increasing doses. As a result of the decrease in the volume of distribution, the terminal disposition half-life at 30 mg/kg was significantly decreased. However, the decreases in the volume of distribution and terminal disposition half-life are of limited clinical importance, since steady-state trough concentrations in serum increase in proportion to dose. Combined results of all multiple-dose studies with similar durations of sample collection indicate no dose-related differences for any pharmacokinetic parameters from 3 to 12 mg/kg. As observed in the present study, no dose-related differences exist for teicoplanin total and renal clearances from 3 to 30 mg/kg. However, at 30 mg/kg, a significant decrease in the steady-state volume of distribution is observed. As a consequence of the reduction in the volume of distribution at 30 mg/kg with no change in clearance, the terminal disposition half-life is decreased.

Pooling data for stability studies: testing the equality of batch degradation slopes.

Pharmaceutical products are routinely monitored for their stability over time. Stability studies generally consist of a random sample of dosage units (e.g., tablets, capsules, vials) from a batch or several batches placed in a storage room and periodically assayed for their drug content. The degradation of the drug product is modeled, and according to the Guideline for Submitting Documentation for Stability Studies of Human Drugs and Biologics (Food and Drug Administration, 1987), the shelf-life is calculated as the time point at which the lower 95% confidence limit about the fitted regression line crosses the lowest acceptable limit for drug content (frequently 90% of the labeled amount). When multiple batches are manufactured, preliminary testing for any batch differences (both slope and intercept) should precede pooling stability data from all batches in the analysis. The Guideline recommends a level of significance of .25 for such preliminary testing based on the work described by Bancroft (1964, Biometrics 20, 427-442). Using such a large significance level helps ensure that the power of the test for the batch differences is sufficiently high. This paper presents an approach whereby the power of the test is fixed and the significance level of the test needed to obtain this power is calculated from the data. If the observed significance level does not achieve the calculated significance level, then the data can be pooled. Examples will illustrate the relative performance of the FDA guideline and the proposed procedure.

Comparison of vancomycin- and teicoplanin-induced histamine release and "red man syndrome".

Twelve healthy adult males participated in a double-blind, randomized, two-way crossover study to determine histamine release and the frequency and severity of "red man syndrome" (RMS) following intravenous administration of vancomycin (15 mg/kg of body weight over 60 min) and teicoplanin (15 mg/kg over 30 min). Concentrations of vancomycin and teicoplanin in serum and concentrations of histamine in plasma were measured at baseline and during and after each infusion. Erythema and pruritus were classified a priori as mild, moderate, or severe. The extent of erythema was determined by the use of a burn chart, and pruritus was assessed by the subject with a rank scale. Global severity of RMS was determined by summation of the individual scores for pruritus and erythema. Baseline areas under the concentration-time curve for histamine were not significantly different for the vancomycin and teicoplanin treatments. Vancomycin caused RMS in 11 of 12 subjects (9 severe and 2 moderate cases) and was associated with a significant increase in plasma histamine (46.7 +/- 31.3 ng.min/ml, P less than 0.05). In contrast, teicoplanin did not cause RMS or elicit significant histamine release (8.7 +/- 13.2 ng.min/ml). Peak concentrations of vancomycin and teicoplanin in serum were 58.8 +/- 8.4 and 148.0 +/- 31.8 micrograms/ml, respectively (P less than 0.05). Assuming equal efficacy, these data suggest that teicoplanin may be a safe alternative agent in subjects experiencing severe RMS due to vancomycin; however, further studies in the clinical setting are needed.