Proof-of-concept study on the suitability of 13C-urea as a marker substance for assessment of in vivo behaviour of oral colon-targeted dosage forms

Background and purpose:

¹³C-urea may be a suitable marker to assess the in vivo fate of colon-targeted dosage forms given by mouth. We postulated that release in the colon (urease-rich segment) of ¹³C-urea from colon-targeted capsules would lead to fermentation of ¹³C-urea by bacterial ureases into ¹³CO₂. Subsequent absorption into the blood and circulation would lead to detectable ¹³C (as ¹³CO₂) in breath. If, however, release of ¹³C-urea occurred in the small intestine (urease-poor segment), we expected detectable ¹³C (as ¹³C-urea) in blood but no breath ¹³C (as ¹³CO₂). The differential kinetics of ¹³C-urea could thus potentially describe both release kinetics and indicate the gastrointestinal segment of release.

Experimental approach:

The in vivo study consisted of three experiments, during which the same group of four volunteers participated.

Key results:

The kinetic model was internally valid. The appearance of ¹³C-in breath CO₂ (F_fermented) and the appearance of ¹³C in blood as ¹³C-urea (F_{not fermented}) show a high inverse correlation (Pearson''s r=−0.981, P= 0.06). The total recovery of ¹³C (F_fermented+F_{not fermented}) averaged 99%, indicating complete recovery of the administered ¹³C via breath and blood. ¹³CO₂ exhalation was observed in all subjects. This indicates that ¹³C-urea was available in urease-rich segments, such as the caecum or colon.

Conclusions and implications:

In this proof-of-concept study, ¹³C-urea was able to provide information on both the release kinetics of a colon-targeted oral dosage form and the gastrointestinal segment where it was released.     

Association of cervical cancer with the presence of CD4+ regulatory T cells specific for human papillomavirus antigens

Because of their important role in the maintenance of self-tolerance, CD4(+) regulatory T cells prevent autoimmune diseases but also curtail the efficacy of T cell immune responses against cancers. We now show that this suppressive action of CD4(+) regulatory T cells is not limited to cancers displaying tumor-associated self antigens, such as melanomas, but also extends to human papillomavirus (HPV)-positive cervical cancers that express foreign tumor antigens. HPV-specific CD4(+) T cells isolated from lymph node biopsies of cervical cancer patients were found to suppress proliferation and cytokine (IFN-gamma, IL-2) production by responder T cells. The capacity of HPV-specific CD4(+) T cells to exert this suppressive effect depended on their activation by cognate HPV antigen and on close-range interactions with responder T cells. HPV-specific CD4(+) regulatory T cells were also retrieved from cervical cancer biopsies, suggesting that they interfere with the anti-tumor immune response at both the induction and effector levels. Our findings offer a plausible explanation for the observed failure of the tumor-specific immune response in patients with cervical carcinoma.     

Distinct regulation and impact of type 1 T-cell immunity against HPV16 L1, E2 and E6 antigens during HPV16-induced cervical infection and neoplasia

Cervical cancer is the possible outcome of a genital infection with high-risk human papillomavirus type 16 (HPV16) and is preceded by a phase of persistent HPV infection during which the host immune system fails to eliminate the virus. Our previous work showed that failure is reflected by the absence of type 1 T-cell immunity against HPV16 early antigens E2 and E6 in patients with HPV16+ cervical lesions. We now show that a majority of both patients with cervical lesions and healthy subjects display HPV16 L1 peptide-specific type 1 T-cell responses with similar magnitude. The T-cell response in patients was directed at a broad range of peptides within L1, suggesting that during persistent or repeated exposure to HPV16 L1, the immune system maximizes its efforts to counter the viral challenge. Unlike the type 1 T-cell responses against HPV16 early antigens E2 and E6, type 1 T-cell immunity against L1 does not correlate with health or disease. This argues that T-cell responses against early and late HPV16 antigens essentially differ in the manner in which they are induced and regulated, as well as in their impact on the subsequent stages of HPV16-induced cervical disease.     

Detection of human papillomavirus type 18 E6 and E7-specific CD4+ T-helper 1 immunity in relation to health versus disease

The most common high-risk human papillomavirus types, HPV16 and 18, differ markedly with respect to their interaction with the host. Clearance of HPV18 infections generally takes longer and HPV18-positive cancers have a poorer prognosis. We therefore evaluated Th1-type immunity against the E6 and E7 oncoproteins of HPV18 in healthy subjects and in patients with HPV18-positive genital cancer, and compared the results to our previously obtained data for HPV16. Approximately 20% of the healthy individuals displayed immunity against HPV18 E6. In contrast, none of the patients showed such responses, despite the presence of HPV18-positive lesions. Several of the patients did respond to HPV18 E7, whereas this immunity is rarely found in healthy subjects. This pattern of immune reactivity is essentially similar to that previously found for HPV16. It is unlikely that this similarity is the result of immunological cross-reactivity between the E6 and E7 antigens of HPV types 16 and 18. Our data confirm the relation between failure of E6-specific Th1 immunity and high-risk HPV-induced cervical neoplasia and argue that parameters other than these determine the differences in pathological impact between HPV types 16 and 18.     

Bipolar and monopolar radiofrequency treatment of osteoarthritic knee articular cartilage: acute and temporal effects on cartilage compressive stiffness, permeability, cell synthesis, and extracellular matrix composition

The cellular, biochemical, biomechanical, and histologic effects of radiofrequency-generated heat on osteoarthritic cartilage were assessed. Articular cartilage explants (n=240) from 26 patients undergoing total knee arthroplasty were divided based on Outerbridge grade (I or II/III) and randomly assigned to receive no treatment (controls) or monopolar or bipolar radiofrequency at 15 or 30 W. Both potentially beneficial and harmful effects of radiofrequency treatment of articular cartilage were noted. It will be vital to correlate data from in vitro and in vivo study of radiofrequency thermal chondroplasty to determine the clinical usefulness of this technique.     

Frequent display of human papillomavirus type 16 E6-specific memory t-Helper cells in the healthy population as witness of previous viral encounter

Genital human papillomavirus (HPV) infection is common and the majority of infected individuals successfully deal with this virus. Clearance of HPV is presumably mediated by T cells but HPV-16-specific T-cell memory was usually detected in patients with progressive disease and not in healthy subjects, suggesting that HPV-immunity comes too late. We now show the presence of HPV-16 E6-specific memory T-helper (Th) responses in a major fraction (12 of 20) of healthy individuals by application of the IFN-gamma-ELISPOT assay. Although nearly all E6-peptides were recognized, the majority of the responders targeted peptide sequences of the COOH-terminal half (E6(81-158)) of HPV-16 E6. In a direct comparison, the presence of HPV-16 E6-specific T cells coincided with HPV-16 E2-specific T-cell reactivity in healthy individuals, whereas hardly any HPV-16 E7-specific Th immunity was found. This indicates that the induction of T-cell reactivity against HPV-16 E7 is suboptimal during infection when compared with that against HPV-16 E2 and HPV-16 E6. In conclusion, the presence of HPV-16 E6-specific Th memory in the healthy population demonstrates that HPV infection leads to T-cell immunity against immediate early proteins expressed during infection. Because this HPV-16 E6-specific T-cell immunity was frequently detected in healthy subjects, our data suggest that the observed IFN-gamma-producing proliferating T cells circulating in the peripheral blood play a role in protection against persistent HPV infection and associated development of malignancies.     

Alteration in methyl-methanesulfonate-induced poly(ADP-ribosyl)ation by 2-butoxyethanol in Syrian hamster embryo cells

The effects of 2-butoxyethanol (2-BE) on poly(ADP-ribosyl)ation were studied in Syrian hamster embryo (SHE) cells by measuring the cellular concentrations of the polymer poly(ADP-ribose) (pADPr) and of NAD+, the substrate of poly(ADP-ribose) polymerase (PARP). As biotransformation pathways of ethylene glycol ethers involve NAD+-dehydrogenases, it was hypothesized that 2-BE could reduce poly(ADP-ribosyl)ation by consuming NAD+. As a result DNA repair could be altered, which would explain that 2-BE had been shown to potentiate the effects of clastogenic substances such as methyl-methanesulfonate (MMS). In this study, the effects of 2- BE on MMS-induced pADPr metabolism were analyzed. The results indicated that: (i) 2-BE (5 mM) by itself did not influence significantly pADPr or NAD+ levels. (ii) 2-BE inhibited pADPr synthesis in MMS (0.2 mM)- pretreated cells, without any change in NAD+ concentrations. (iii) MMS treatment, which rapidly increased pADPr levels, also affected the poly(ADP-ribosyl)ation system as a secondary effect by damaging cell structures. Membrane permeabilization, which occurred at concentrations >1 mM MMS, led to a dramatic leakage of cellular NAD+ resulting in a strong reduction in pADPr levels. (iv) A bleomycin pulse (100 microM) applied after MMS and/or 2-BE treatment confirmed that 2-BE reduced poly(ADP-ribosyl)ation capacities of MMS-treated cells, though the glycol ether had no effect alone. This study confirmed that the inhibition of pADPr synthesis could be responsible for the synergistic effects of 2-BE with genotoxic substances. The mechanism of this inhibition cannot be explained by a lack of NAD+ at the concentrations of 2-BE tested.      

Local metabolism in lung airways increases the uncertainty of pyrene as a biomarker of polycyclic aromatic hydrocarbon exposure

While inhaled polycyclic aromatic hydrocarbons have long been suspected to induce lung cancer in humans, their dosimetry has not been fully elucidated. A key question is whether the critical exposure occurs during absorption in the lungs, or if toxicants in the systemic circulation contribute significantly to lung cancer risk. In particular, data are needed to determine how the physical properties of inhalants affect local dosimetry in the respiratory tract. Pyrene, a tobacco smoke component, was selected for study because it has physical properties between those of highly lipophilic benzo[a]pyrene and water- soluble nitrosamines. Aliquots of 5 ng of pyrene dissolved in a phospholipid/ saline suspension were instilled as a single-spray bolus in the posterior trachea of the dog just anterior to the carina. For 3 h after instillation, blood was repeatedly sampled from the azygous vein, which drains the mucosa around the point of instillation, and from both sides of the systemic circulation. At 3 h post-instillation, tissue samples were taken. Autoradiography was used to determine the depth distribution of pyrene in the tracheal mucosa. The concentration of pyrene-equivalent radioactivity in the azygous vein peaked 9 min after the instillation. At approximately 30 min after instillation, a rapid early clearance phase shifted into a distinctly slower second clearance phase. Rates of rapid clearance were, however, sufficiently slow to indicate diffusion-limited absorption of pyrene in the trachea. This finding was corroborated by high concentrations of pyrene in the epithelium as determined by autoradiography. High epithelial concentration of pyrene combined with a slow penetration into the circulating blood allowed substantial first-pass metabolic conversion of pyrene in the tracheal mucosa. A total of 13% of the instilled pyrene was retained in the tracheal mucosa 3.2 h after instillation; of this, 29% was parent compound, 52% was organic-extractable metabolites, 14% was water-soluble metabolites and 6% (approximately 1% of the instilled amount) was covalently bound to tracheal tissues. Results support the inference that lipophilic protoxicants, because of slow, diffusion-limited absorption, are more likely than water-soluble protoxicants to be bioactivated in the lining epithelium and, in turn, induce first-pass toxicity at the site of entry. In addition, limitations were identified in the use of systemically distributed biomarkers of PAHs, such as urinary hydroxypyrene levels, as indicators of the biologically effective dose in airway target cells.      

Prediction of a mismatch repair gene defect by microsatellite instability and immunohistochemical analysis in endometrial tumours from HNPCC patients

Instability of microsatellite repeat sequences has been observed in colorectal carcinomas and in extracolonic malignancies, predominantly endometrial tumours, occurring in the context of hereditary non-polyposis colorectal cancer (HNPCC). Microsatellite instability (MSI) as a feature of human DNA mismatch repair (MMR)-driven tumourigenesis of the uterine mucosa has been studied primarily in sporadic tumours showing predominantly somatic hypermethylation of MLH1. The present study shows that all endometrial carcinomas (n=12) from carriers of MLH1 and MSH2 germline mutations demonstrate an MSI-high phenotype involving all types of repeat markers, while in endometrial carcinomas from MSH6 mutation carriers, only 36% (4 out of 11) demonstrate an MSI-high phenotype. Interestingly, an MSI-high phenotype was found in endometrial hyperplasias from MSH2 mutation carriers, in contrast to hyperplasias from MLH1 mutation carriers, which exhibited an MSI-stable phenotype. Instability of only mononucleotide repeat markers was found in both endometrial carcinomas and hyperplasias from MSH6 mutation carriers. In 29 out of 31 (94%) endometrial tumour foci, combined MSI and immunohistochemical analysis of MLH1, MSH2, and MSH6 could predict the identified germline mutation. The observation of MSI in endometrial hyperplasia and of altered protein staining for the MMR genes supports the idea that inactivation of MMR genes is an early event in endometrial tumourigenesis. A correlation was found between the variation in the extent and level of MSI and the age of onset of carcinoma, suggesting differences in the rate of tumour progression. A high frequency of MSI in hyperplasias, found only in MSH2 mutation carriers, might indicate a more rapid tumour progression, correlating with an earlier age of onset of carcinoma. The present study indicates that assessment of altered protein staining combined with MSI analysis of endometrial tumours might direct the mutational analysis of MMR genes.