Enantiomeric profiling of a chemically diverse mixture of chiral pharmaceuticals in urban water.

Due to concerns regarding the release of pharmaceuticals into the environment and the understudied impact of stereochemistry of pharmaceuticals on their fate and biological potency, we focussed in this paper on stereoselective transformation pathways of selected chiral pharmaceuticals (16 pairs) at both microcosm (receiving waters and activated sludge wastewater treatment simulating microcosms) and macrocosm (wastewater treatment plant (WWTP) utilising activated sludge technology and receiving waters) scales in order to test the hypothesis that biodegradation of chiral drugs is stereoselective. Our monitoring programme of a full scale activated sludge WWTP and receiving environment revealed that several chiral drugs, those being marketed mostly as racemates, are present in wastewater and receiving waters enriched with one enantiomeric form (e.g. fluoxetine, mirtazapine, salbutamol, MDMA). This is most likely due to biological metabolic processes occurring in humans and other organisms. Both activated sludge and receiving waters simulating microcosms confirmed our hypothesis that chiral drugs are subject to stereoselective microbial degradation. It led, in this research, to preferential degradation of S-(+)-enantiomers of amphetamines, R-(+)-enantiomers of beta-blockers and S-(+)-enantiomers of antidepressants. In the case of three parent compound - metabolite pairs (venlafaxine - desmethylvenlafaxine, citalopram - desmethylcitalopram and MDMA - MDA), while parent compounds showed higher resistance to both microbial metabolism and photodegradation, their desmethyl metabolites showed much higher degradation rate both in terms of stereoselective metabolic and non-stereoselective photochemical processes. It is also worth noting that metabolites tend to be, as expected, enriched with enantiomers of opposite configuration to their parent compounds, which might have significant toxicological consequences when evaluating the metabolic residues of chiral pollutants.

Anti-inflammatory effects of infliximab in mice are independent of tumour necrosis factor α neutralization.

Infliximab (IFX) has been used repeatedly in mouse preclinical models with associated claims that anti-inflammatory effects are due to inhibition of mouse tumour necrosis factor (TNF)-α. However, the mechanism of action in mice remains unclear. In this study, the binding specificity of IFX for mouse TNF-α was investigated ex vivo using enzyme-linked immunosorbent assay (ELISA), flow cytometry and Western blot. Infliximab (IFX) did not bind directly to soluble or membrane-bound mouse TNF-α nor did it have any effect on TNF-α-induced nuclear factor kappa B (NF-κB) stimulation in mouse fibroblasts. The efficacy of IFX treatment was then investigated in vivo using a TNF-α-independent Trichuris muris-induced infection model of chronic colitis. Infection provoked severe transmural colonic inflammation by day 35 post-infection. Colonic pathology, macrophage phenotype and cell death were determined. As predicted from the in-vitro data, in-vivo treatment of T. muris-infected mice with IFX had no effect on clinical outcome, nor did it affect macrophage cell phenotype or number. IFX enhanced apoptosis of colonic immune cells significantly, likely to be driven by a direct effect of the humanized antibody itself. We have demonstrated that although IFX does not bind directly to TNF-α, observed anti-inflammatory effects in other mouse models may be through host cell apoptosis. We suggest that more careful consideration of xenogeneic responses should be made when utilizing IFX in preclinical models.

Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility.

Cellular adaptation to hypoxia occurs via a complex programme of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1ß and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity.

Inflammasome-dependent IL-1ß release depends upon membrane permeabilisation.

Interleukin-1ß (IL-1ß) is a critical regulator of the inflammatory response. IL-1ß is not secreted through the conventional ER-Golgi route of protein secretion, and to date its mechanism of release has been unknown. Crucially, its secretion depends upon the processing of a precursor form following the activation of the multimolecular inflammasome complex. Using a novel and reversible pharmacological inhibitor of the IL-1ß release process, in combination with biochemical, biophysical, and real-time single-cell confocal microscopy with macrophage cells expressing Venus-labelled IL-1ß, we have discovered that the secretion of IL-1ß after inflammasome activation requires membrane permeabilisation, and occurs in parallel with the death of the secreting cell. Thus, in macrophages the release of IL-1ß in response to inflammasome activation appears to be a secretory process independent of nonspecific leakage of proteins during cell death. The mechanism of membrane permeabilisation leading to IL-1ß release is distinct from the unconventional secretory mechanism employed by its structural homologues fibroblast growth factor 2 (FGF2) or IL-1α, a process that involves the formation of membrane pores but does not result in cell death. These discoveries reveal key processes at the initiation of an inflammatory response and deliver new insights into the mechanisms of protein release.

Quantitative dynamic imaging of immune cell signalling using lentiviral gene transfer.

Live-cell imaging of fluorescent fusion proteins has transformed our understanding of mammalian cell signalling and function. However, some cellular systems such as immune cells are unsuitable or refractory to many existing transgene delivery methods thus limiting systematic analyses. Here, a flexible lentiviral gene transfer platform for dynamic time-lapse imaging has been developed and validated with single-molecule spectroscopy, mathematical modelling and transcriptomics and used for analysis of a set of inflammation-related signalling networks. Time-lapse imaging of nuclear factor kappa B (NF-κB), signal transducer and activator of transcription (STATs) and nuclear factor of activated T-cells (NFAT) in mammalian immune cell lines provided evidence for heterogeneous temporal encoding of inflammatory signals. In particular, the absolute quantification of single-cell responses over time via fluorescent correlation spectroscopy (FCS) showed that NF-κB p65 activation in response to tumour necrosis factor α (TNFα) was differentially encoded in variable amplitude of nuclear translocation between immune and non-immune cells. The absolute number of activated molecules was dictated in part by the cell size, suggesting a morphology-dependent regulatory mechanism. The developed platform will enable further absolute quantitative analyses of the dynamic interactions between signalling networks, in and between individual cells, allowing better integration with mathematical models of signalling networks.

Using chiral liquid chromatography quadrupole time-of-flight mass spectrometry for the analysis of pharmaceuticals and illicit drugs in surface and wastewater at the enantiomeric level.

This paper presents and compares for the first time two chiral LC-QTOF-MS methodologies (utilising CBH and Chirobiotic V columns with cellobiohydrolase and vancomycin as chiral selectors) for the quantification of amphetamine, methamphetamine, MDA (methylenedioxyamphetamine), MDMA (methylenedioxymethamphetamine), propranolol, atenolol, metoprolol, fluoxetine and venlafaxine in river water and sewage effluent. The lowest MDLs (0.3-5.0 ng L(-1) and 1.3-15.1 ng L(-1) for river water and sewage effluent respectively) were observed using the chiral column Chirobiotic V. This is with the exception of methamphetamine and MDMA which had lower MDLs using the CBH column. However, the CBH column resulted in better resolution of enantiomers (R(s)=2.5 for amphetamine compared with R(s)=1.2 with Chirobiotic V). Method recovery rates were typically >80% for both methodologies. Pharmaceuticals and illicit drugs detected and quantified in environmental samples were successfully identified using MS/MS confirmation. In sewage effluent, the total beta-blocker concentrations of propranolol, atenolol and metoprolol were on average 77.0, 1091.0 and 3.6 ng L(-1) thus having EFs (Enantiomeric Fractions) of 0.43, 0.55 and 0.54 respectively. In river water, total propranolol and atenolol was quantified on average at <10.0 ng L(-1). Differences in EF between sewage and river water matrices were evident: venlafaxine was observed with respective EF of 0.43 ± 0.02 and 0.58 ± 0.02.

Resource dependent biodegradation of estrogens and the role of ammonia oxidising and heterotrophic bacteria.

The influence of ammonia oxidising bacteria and bulk organic competition was assessed during laboratory scale activated sludge treatment. Under short and long hydraulic retention time (HRT) and solid retention time (SRT) conditions, bioreactors were supplied with synthetic sewage spiked with 0.04-2.1 mg m(3) d(-1) of steroid estrogens with and without ammonia as a nitrogen source. Non acclimated biomass that had previously not been exposed to estrogens was capable of biodegrading estrogens (89% and 78%) within 24 h in the short HRT/SRT and long HRT/SRT conditions respectively. Changing the nitrogen source from ammonia to nitrate caused reductions in ammonia oxidising bacteria (AOB) numbers from 2.47×10(8) to 1.17×10(7)AOB mL(-1) and 5.15×10(9) to 4.27×10(7)AOB mL(-1) for the short and long HRT/SRT conditions respectively. Despite these reductions, biodegradation of estrogens was unaffected, which demonstrated that heterotrophic bacteria were able to biodegrade estrogens. Estrogen biodegradation was unrestricted and estrogen could be removed at higher than environmental concentrations following a pseudo-first order relationship. During this study, bulk organic loading appeared not to have any appreciable influence upon estrogen biodegradation. These results suggest heterotrophic bacteria, capable of scavenging a broad spectrum of organic material, carry out estrogen biodegradation.

Evaluation of the impact of intraobserver variability on endometrial dating and the diagnosis of luteal phase defects.

OBJECTIVE: To determine the magnitude of intraobserver variation in dating endometrial biopsies and its impact on clinical management. DESIGN: Blinded histopathologic interpretation of endometrial biopsy specimens 1 year apart by five pathologists. SETTING: Large military tertiary care center. PATIENTS: Endometrial biopsy specimens from 51 patients undergoing evaluation for potential luteal phase defects. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Calculation of the magnitude of the individual and overall intraobserver variation in endometrial dating for the five pathologists and estimation of its potential impact on clinical management. RESULTS: The intraobserver variation was 0.69 +/- 0.05 days (means +/- SE). There was no significant difference in the magnitude of the variation for 1-day or 2-day dating ranges. The theoretical probability of altering clinical management by having the same pathologist redate a given specimen ranged from 15% to 28%. CONCLUSION: Histologic dating of endometrial biopsies is subject to a small but highly clinically significant intraobserver variability that may have a major impact on clinical management.

Synthesis, properties, and intratumoral evaluation of mitoxantrone-loaded casein microspheres in Lewis lung carcinoma.

Smooth, round, uniform bovine casein microspheres of 1-5 and 10-20 microns size were readily prepared by a steric stabilization technique previously developed in this laboratory for synthesis of albumin microspheres. The avid phagocytic uptake of casein and albumin microspheres was demonstrated with fluorescein-labelled microspheres using a macrophage-like mouse myelomonocytic leukaemia cell line. Post-synthesis loading of 25% mitoxantrone was achieved for casein microspheres containing 20% polyglutamic acid. Preliminary intratumoural chemotherapy experiments with a mouse Lewis lung carcinoma indicated that mitoxantrone and mitoxantrone-loaded casein-polyglutamic acid microspheres exhibited lower toxicity when administered intratumorally.

The effect of interobserver variation in dating endometrial histology on the diagnosis of luteal phase defects.

Endometrial biopsy specimens (n = 62) were evaluated by five pathologists to assess the effect of interobserver variation on histologic dating of the endometrium. The potential effect of this variation on the diagnosis of luteal phase defects (LPDs) and resulting clinical management was also determined. Mean (+/- standard error) interobserver variation was 0.96 +/- 0.08 days, comparable to results reported by other investigators. The magnitude of the variation was not affected by whether the biopsy specimen was obtained in the mid or late luteal phase, the degree of lag between the dating and subsequent menses, or the presence of an LPD. Redating of a specimen by another pathologist would have resulted in a change in the determination of "in" or "out" of phase in 22% of cases. The subsequent probability of changing patient management altered ranged from 22% to 39% depending on the clinical setting.

Biopsy evaluation of chronic active hepatitis. Peritoneoscopy with directed liver biopsy versus blind percutaneous liver biopsy.

In this prospective study of chronic active liver disease, we compared the assessment of hepatic histology in samples obtained by peritoneoscopy with directed liver biopsy and blind percutaneous liver biopsy in 23 cases (22 patients, one patient studied twice). In blinded fashion, a pathologist assessed all specimens for evidence of cirrhosis and degree of necroinflammatory change. Two clinicians independently reviewed clinical and laboratory findings in both sets of biopsies. Each committed in writing recommendations regarding immunosuppressive therapy, follow-up interval, and rebiopsy date. The final diagnosis differed from that made by percutaneous and directed biopsy in 2 of 23 (9%) and 1 of 23 (4%) cases, respectively. Six cases of cirrhosis were correctly diagnosed by both biopsy methods, but only four of the six cirrhotic cases were diagnosed by gross peritoneoscopic findings. In only 2 of 23 (9%) cases was there disagreement in the degree of necroinflammatory change between the blind and directed biopsies that affected treatment recommendations. We conclude that blind percutaneous biopsy adequately diagnoses and monitors activity in viral chronic hepatitis for treatment purposes.

Technetium-99m red blood cell blood-pool imaging in functional asplenia due to leukemic infiltration.

Tc-99m labeled RBC blood-pool and Tc-99m sulfur colloid imaging were performed in a patient with chronic myelogenous leukemia (CML) in blast crisis. Functional asplenia was demonstrated on the sulfur colloid study despite intact organ perfusion as demonstrated by blood-pool imaging. Tc-99m RBC blood-pool imaging appears to be an ideal noninvasive technique to document intact spleen perfusion in functional asplenia.

Histiocytoid carcinoma: a variant of breast cancer.

The history of a patient presenting with metachronous bilateral breast cancer displaying histiocytoid features is reviewed. Although regional metastases were noted, this patient has not demonstrated an aggressive systemic disease pattern. In the past, histiocytoid breast cancer has been classified as either a lipid-rich carcinoma or as a variant of lobular carcinoma. However, histiocytoid carcinoma should be considered a distinct entity. Unlike the lipid-rich carcinomas, this tumor stained strongly for mucin. Immunoperoxidase staining indicated strong positively for CEA and negative staining for alpha-lactalbumin. There is suggestive evidence of a relationship between histiocytoid breast carcinoma and breast cancers of apocrine origin. Controversy remains and further evaluation is needed to elucidate the histiogenesis and biological potential of this neoplasm.

Development of nongerm cell malignancies in nonseminomatous germ cell tumors.

We report 2 cases of sarcomatous transformation of mixed germ cell tumors of the testis. Both patients received cisplatin-based combination chemotherapy. Embryonal rhabdomyosarcoma, chondrosarcoma and spindle cell sarcoma were the dominant pathological patterns in the metastatic sites. Nongerm cell malignancies in mixed germ cell tumors following chemotherapy may be more common than previously described.

Autoradiographic localisation of opiate receptors in rat small intestine.

[3H]Naloxone and [3H]dihydromorphine are selective ligands for opiate receptors. Using an in vitro autoradiographic technique, binding of these ligands has been demonstrated to the villi and crypts in rat small intestine. These results indicate the presence of opiate receptor sites in the small intestine which suggests further a role for endogenous opiates in the transport functions of intestinal mucosa.

Acute rheumatic carditis causing sudden death.

An 18-year-old male died suddenly while running a confidence course in basic training. Past medical history was negative for acute rheumatic fever. At autopsy he had acute rheumatic mitral valvulitis with extensive myocarditis. Multiple Aschoff bodies were seen in perivascular regions in the right and left ventricle. Review of recent literature of the various causes of sudden cardiac death failed to reveal acute rheumatic valvulitis and myocarditis as reported causes of sudden death.

Paratesticular mesotheliomas.

Paratesticular mesotheliomas are uncommon tumors. We report 2 such cases and discuss their clinical and pathological findings.

An autoradiographic demonstration of prazosin binding to arterial vessels in the rat.

Prazosin is believed to have an antihypertensive action due to a selective blockade of alpha-adrenoceptors on vascular smooth muscle. Using an in vitro autoradiographic technique specific binding of [3H]prazosin to the abdominal aorta and renal artery in the rat has been shown. The binding of [3H]prazosin to arterial vessels may provide some evidence for the site where prazosin exerts its antihypertensive action.