In pursuit of the triple crown: mechanism-based pharmacodynamic modelling for the optimization of three-drug combinations against KPC-producing Klebsiella pneumoniae.

OBJECTIVES: Optimal combination therapy for Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) is unknown. The present study sought to characterize the pharmacodynamics (PD) of polymyxin B (PMB), meropenem (MEM) and rifampin (RIF) alone and in combination using a hollow fibre infection model (HFIM) coupled with mechanism-based modelling (MBM). METHODS: A 10-day HFIM was utilized to simulate human pharmacokinetics (PK) of various PMB, MEM and RIF dosing regimens against a clinical KPC-Kp isolate, with total and resistant subpopulations quantified to capture PD response. A MBM was developed to characterize bacterial subpopulations and synergy between agents. Simulations using the MBM and published population PK models were employed to forecast the bacterial time course and the extent of its variability in infected patients for three-drug regimens. RESULTS: In the HFIM, a PMB single-dose ('burst') regimen of 5.53 mg/kg combined with MEM 8 g using a 3-hr prolonged infusion every 8 hr and RIF 600 mg every 24 hr resulted in bacterial counts below the quantitative limit within 24 hr and remained undetectable throughout the 10-day experiment. The final MBM consisted of two bacterial subpopulations of differing PMB and MEM joint susceptibility and the ability to form a non-replicating, tolerant subpopulation. Synergistic interactions between PMB, MEM and RIF were well quantified, with the MBM providing adequate capture of the observed data. DISCUSSION: An in vitro-in silico approach answers questions related to PD optimization as well as overall feasibility of combination therapy against KPC-Kp, offering crucial insights in the absence of clinical trials.

Using machine learning to optimize antibiotic combinations: dosing strategies for meropenem and polymyxin B against carbapenem-resistant Acinetobacter baumannii.

OBJECTIVES: Increased rates of carbapenem-resistant strains of Acinetobacter baumannii have forced clinicians to rely upon last-line agents, such as the polymyxins, or empirical, unoptimized combination therapy. Therefore, the objectives of this study were: (a) to evaluate the in vitro pharmacodynamics of meropenem and polymyxin B (PMB) combinations against A. baumannii; (b) to utilize a mechanism-based mathematical model to quantify bacterial killing; and (c) to develop a genetic algorithm (GA) to define optimal dosing strategies for meropenem and PMB. METHODS: A. baumannii (N16870; MICmeropenem = 16 mg/L, MICPMB = 0.5 mg/L) was studied in the hollow-fibre infection model (initial inoculum 108 cfu/mL) over 14 days against meropenem and PMB combinations. A mechanism-based model of the data and population pharmacokinetics of each drug were used to develop a GA to define the optimal regimen parameters. RESULTS: Monotherapies resulted in regrowth to ~1010 cfu/mL by 24 h, while combination regimens employing high-intensity PMB exposure achieved complete bacterial eradication (0 cfu/mL) by 336 h. The mechanism-based model demonstrated an SC50 (PMB concentration for 50% of maximum synergy on meropenem killing) of 0.0927 mg/L for PMB-susceptible subpopulations versus 3.40 mg/L for PMB-resistant subpopulations. The GA had a preference for meropenem regimens that improved the %T > MIC via longer infusion times and shorter dosing intervals. The GA predicted that treating 90% of simulated subjects harbouring a 108 cfu/mL starting inoculum to a point of 100 cfu/mL would require a regimen of meropenem 19.6 g/day 2 h prolonged infusion (2 hPI) q5h + PMB 5.17 mg/kg/day 2 hPI q6h (where the 0 h meropenem and PMB doses should be 'loaded' with 80.5% and 42.2% of the daily dose, respectively). CONCLUSION: This study provides a methodology leveraging in vitro experimental data, a mathematical pharmacodynamic model, and population pharmacokinetics provide a possible avenue to optimize treatment regimens beyond the use of the 'traditional' indices of antibiotic action.

Enabling dual cellular destinations of polymeric nanoparticles for treatment following partial injury to the central nervous system.

Following neurotrauma, oxidative stress is spread via the astrocytic syncytium and is associated with increased aquaporin 4 (AQP4), inflammatory cell infiltration, loss of neurons and glia and functional deficits. Herein we evaluate multimodal polymeric nanoparticles functionalized with an antibody to an extracellular epitope of AQP4, for targeted delivery of an anti-oxidant as a therapeutic strategy following partial optic nerve transection. Using fluorescence microscopy, spectrophotometry, correlative nanoscale secondary ion mass spectrometry (NanoSIMS) and transmission electron microscopy, in vitro and in vivo, we demonstrate that functionalized nanoparticles are coated with serum proteins such as albumin and enter both macrophages and astrocytes when administered to the site of a partial optic nerve transection in rat. Antibody functionalized nanoparticles synthesized to deliver the antioxidant resveratrol are effective in reducing oxidative damage to DNA, AQP4 immunoreactivity and preserving visual function. Non-functionalized nanoparticles evade macrophages more effectively and are found more diffusely, including in astrocytes, however they do not preserve the optic nerve from oxidative damage or functional loss following injury. Our study highlights the need to comprehensively investigate nanoparticle location, interactions and effects, both in vitro and in vivo, in order to fully understand functional outcomes.

Unraveling the relationship between structure and stabilization of triarylpyridines as G-quadruplex binding ligands.

A series of novel 2,4,6-triarylpyridines have been synthesized and their interactions with intramolecular G-quadruplexes have been measured by Förster Resonance Energy Transfer (FRET) melting and Fluorescent Intercalator Displacement (FID) assays. A few of these compounds exhibit stabilization of G4-DNA that is comparable to other benchmark G4-DNA ligands with fair to excellent G4-DNA vs. duplex selectivity and significant cytotoxicity towards HeLa cells. The nature of the 4-aryl substituents along with side chain length governs the G4-DNA stabilization ability of the compounds. In addition, we demonstrate that there is a strong correlation between the ability of the compounds to stabilize the same G4-DNA sequence in K(+) and Na(+) conditions and a strong correlation between the ability of the compounds to stabilize different G4-DNA sequences in K(+) or Na(+) buffer.

A microfluidic platform to synthesize a G-quadruplex binding ligand.

An aromatic triarylpyridine chromophore promotes pi-stacking interactions with the terminal G-tetrad in quadruplex DNA, stabilizing the structure and presenting a pathway towards cancer treatment by inhibition of telomerase. An interesting parent compound in this class is the dimethylamino functionalised 4'-aryl-2,6-bis(4-aminophenyl)pyridine. However, access to this compound using traditional batch synthetic methodology is limited, due to thermodynamic and kinetic constraints. A novel approach to the synthesis of this compound has been developed, involving dynamic thin films, overcoming a series of competing reactions, effectively controlling chemical reactivity and selectivity.

Phosphate uptake in chick kidney cells: effects of 1,25(OH)2D3 and 24,25(OH)2D3.

Phosphate homeostasis is controlled in part by absorption from the intestine, and reabsorption in the kidney. While the effect of Vitamin D metabolites on enterocytes is well documented, in the current study we assess selected responses in primary cultures of kidney cells. Time course studies revealed a rapid stimulation of phosphate uptake in cells treated with 1,25(OH)(2)D(3), relative to controls. Dose-response studies indicated a biphasic curve with optimal stimulation at 300 pM 1,25(OH)(2)D(3) and inhibition at 600 pM seco-steroid. Antibody 099--against the 1,25D(3)-MARRS receptor - abolished stimulation by the steroid hormone. Moreover, phosphate uptake was mediated by the protein kinase C pathway. The metabolite 24,25(OH)(2)D(3), which was found to inhibit the rapid stimulation of phosphate uptake in intestinal cells, had a parallel effect in cultured kidney cells. Finally, the 24,25(OH)(2)D(3) binding protein, catalase, was assessed for longer term down regulation. In both intestinal epithelial cells and kidney cells incubated with 24,25(OH)(2)D(3) for 5-24h, both the specific activity of the enzyme and protein levels were decreased relative to controls, while 1,25(OH)(2)D(3) increased both parameters over the same time periods. We conclude that the Vitamin D metabolites have similar effects in both kidney and intestine, and that 24,25(OH)(2)D(3) may have effects at the level of gene expression.

Rapidly increasing prevalence of eosinophilic oesophagitis in Western Australia.

AIM: To assess the prevalence of eosinophilic oesophagitis in a tertiary paediatric gastroenterology clinic population. METHODS: A retrospective audit of Western Australian children investigated for oesophageal disease by paediatric gastroenterologists in the years 1995, 1999 and 2004. Macroscopic appearance of the oesophagus at endoscopy, original histological findings and diagnosis were recorded for each child. Biopsy specimens were blindly re-evaluated, with re-coded histological diagnoses compared with original reports. Age, sex and socioeconomic status were identified for each child. RESULTS: The prevalence of eosinophilic oesophagitis in Western Australia increased over the decade 1995-2004, rising from 0.05 to 0.89 per 10 000 children, with a concomitant increase in the severity of oesophagitis as determined by inflammatory cell numbers and associated features of inflammation. Children diagnosed with eosinophilic oesophagitis had a median age of 78.9 months (6.58 years), with no associated predisposition by sex or socioeconomic status trend. Almost one third of cases were macroscopically normal at endoscopy. All children with an original diagnosis of eosinophilic oesophagitis had > or =40 eosinophils per high-power field. CONCLUSION: Over the decade 1995-2004, a true increase was seen in the prevalence of eosinophilic oesophagitis, not accounted for by diagnostic shift. Histological samples should be taken at endoscopy to confirm or exclude the diagnosis of eosinophilic oesophagitis.

The use of complementary medicines by those with asthma.

Complementary therapies attract considerable media attention and previous surveys of members of an asthma patient organisation suggested that their use by those with asthma was commonplace. This report concerns a study of a stratified cross section of the asthma population designed to give a more representative insight into current usage of complementary therapies. A sift questionnaire was used to identify those with asthma and 785 of those so identified undertook a semi-structured face-to-face interview. Only 6% of the study population were current users of complementary therapies with use being more common amongst those who expressed most concern regarding their current medication. Low use of complementary therapies may well reflect satisfaction with current management and suggests that previous surveys may have been unrepresentative of a more balanced population of those with asthma.

The role of the pathologist in the management of neuroblastoma.

The role of the pathologist in the management of neuroblastoma, in the context of the team approach to these lesions, is discussed. The importance of the provision of fresh material is stressed, and the prognostic importance of histology and ancillary tests is noted. Participation by the team in a children's cancer study group is a vital component of current and future treatment methods.

The effects of overcoming experimental bladder outflow obstruction in fetal sheep.

OBJECTIVE: To develop an ovine model of fetal bladder outflow obstruction and to investigate the effect on the kidney of surgical relief of the obstruction in the prenatal period. METHODS: Ultrasound examination and amniocentesis were performed on 68 date-bred pregnant ewes at day 57 of pregnancy (term = 150 days). Fetal gender was determined using a molecular technique to identify single male fetuses. The urethra and urachus were ligated at hysterotomy on 20 of these fetuses at 75 days' gestation. Comparisons were made with six controls that did not undergo operation. Changes that occurred in fetal urinary tract appearance were detected using serial ultrasound examinations. Seven obstructed cases chosen at random had further prenatal surgery on day 94 to decompress the obstructed urinary tract by vesicostomy. The animals were killed at 110 days' gestation and post-mortem studies were performed. RESULTS: Fourteen days after surgical obstruction, there were increases in the summed renal lengths (33 mm vs. 28 mm, p = 0.003) and renal pelvis anteroposterior (A-P) diameters (8 mm vs. 5.5 mm, p = 0.02). In the group allocated to receive surgical decompression, 9 days' relief of obstruction resulted in significant reductions in summed renal lengths (30 mm vs. 41 mm, p = 0.024; controls 31 mm) and renal pelvis A-P diameters (5.8 mm vs. 8.9 mm, p = 0.012; controls < 2 mm). Postmortem histological examination in the surgical decompression group revealed an estimated number of glomeruli similar to controls and greater than in the obstructed cases. CONCLUSION: Surgical relief of fetal bladder outflow obstruction in ovine mid-pregnancy results in improved renal appearance on ultrasonic and histopathological examinations.

Lethal X-linked microcephaly with dysmorphic features, bilateral optic pathway aplasia and normal eyes.

We describe a family, consisting of two brothers and a maternal uncle who died of an apparently identical condition, within a few days of birth, suggestive of an X-linked mode of inheritance. The propositus (the older sibling) was investigated in detail and showed the following clinical features: microcephaly, facial dysmorphism, malformations of hands and feet, and cryptorchidism. Examination of the brain revealed arhinencephaly, a primitive gyral pattern, arrested cortical maturation, absence of corticofugal tracts and corpus callosum, agenesis of the optic pathway with preserved eyes and oculomotor system, absent auditory pathway, agenesis of the pars compacta of the substantia nigra and severe hypoplasia of the cerebellum and its connections. This family belongs to the group of X-linked microcephalies and has some features in common with the Juberg-Marsidi syndrome. The fact that the CNS abnormalities were incompatible with life and the facial dysmorphic features were quite different makes it unlikely that the affected individuals in this family had Juberg-Marsidi syndrome. However, this does not exclude the possibility that more restricted anterior induction defects may occur in some X-linked microcephalies such as Juberg-Marsidi syndrome resulting in prolonged survival.

Prothrombotic megakaryocyte and platelet changes in hypertension are reversed following treatment: a pilot study.

Platelets are formed from, and their function determined by, bone marrow megakaryocytes (MK). Previous studies have found that hypertension is associated with accentuated platelet function and that some antihypertensive drug classes have antiplatelet activity. We measured MK ploidy (DNA content), size, granularity, and expression of the adhesion molecule glycoprotein (GP) IIIa, using flow cytometry and measures of platelet function, in 12 untreated hypertensive patients and 14 normotensive subjects. Eight hypertensive patients were then treated with losartan (50 mg daily), an angiotensin receptor antagonist that lowers blood pressure, and MK and platelet parameters re-measured after 6 weeks. Hypertensive patients had, as compared with matched normotensive subjects: increased MK ploidy (mean +/- SD) 22.9 +/- 2.2 N versus 20.8 +/- 1.6 N (2P = 0.009); increased platelet size, 10.67 +/- 1.03fl versus 9.26 +/- 0.72fl (2P < 0.001); increased platelet expression of GP IIIa, 108.6 +/- 22.5 versus 92.0 +/- 12.3 (2P = 0.036); and reduced platelet count, (207 +/- 52) x 10(9)/l versus (257 +/- 55) x 10(9)/l (2P = 0.026). Losartan significantly reduced MK ploidy, 22.6 +/- 2.2 N versus 21.4 +/- 1.9 N (2P = 0.006); MK size, 607 +/- 22 versus 579 +/- 16 (2P = 0.003); and lengthened cutaneous bleeding time, 424 +/- 86s versus 563 +/- 164s (2P = 0.011), in hypertensive patients. Losartan did not alter MK granularity or GP IIIa expression, or platelet count, size, mass, GP IIIa expression, or aggregation. The data suggest that platelet changes in hypertension may be secondary to changes in MKs, and that anti-hypertensive treatment can alter MKs and the function of platelets they produce. Since antihypertensive therapy reduces the risk of stroke and myocardial infarction, MKs are a novel therapeutic target for the prevention of vascular events.

Protein solubility and folding monitored in vivo by structural complementation of a genetic marker protein.

Protein misfolding is the basis of a number of human diseases and presents an obstacle to the production of soluble recombinant proteins. We present a general method to assess the solubility and folding of proteins in vivo. The basis of this assay is structural complementation between the alpha- and omega- fragments of beta-galactosidase (beta-gal). Fusions of the alpha-fragment to the C terminus of target proteins with widely varying in vivo folding yield and/or solubility levels, including the Alzheimer's amyloid beta (A beta) peptide and a non-amyloidogenic mutant thereof, reveal an unambiguous correlation between beta-gal activity and the solubility/folding of the target. Thus, structural complementation provides a means of monitoring protein solubility/misfolding in vivo, and should find utility in the screening for compounds that influence the pathological consequences of these processes.

Broadsheet number 56: Mechanisms of fetal loss.

The most significant task of the pathologist examining a fetal death is, if possible, the provision of an explanation for the event, which will allow the clinical attendants to counsel the family in an informed and relevant manner. In some cases, no adequate explanation will be possible, though many conditions will be excluded and the importance of this exercise is worth emphasising. Often, however, a combination of fetal, maternal or placental conditions can be found and a full or partial explanation offered, and it is the purpose of this broadsheet to highlight some of these conditions and provide suggestions as to sources of further practical help.

Unexpected childhood death due to hemolytic uremic syndrome.

Two cases of unexpected childhood death due to hemolytic uremic syndrome are reported. A 21-month-old girl who was discovered dead in bed following a short illness was found at autopsy to have overwhelming sepsis resulting from transmural colitis. Escherichia coli serotype 0157A was isolated from the intestine, and renal changes of hemolytic uremic syndrome were found. A 4-year-old girl died suddenly in hospital from intracranial hemorrhage while being treated for hemolytic uremic syndrome-related renal failure. Culture of urine and feces grew verocytotoxin producing E. coli. These cases demonstrate that hemolytic uremic syndrome may be a rare cause of unexpected childhood death and that the diagnosis may not be established prior to autopsy. Postmortem culture of tissues and fluids in cases of suspected sepsis in children may be essential in establishing this diagnosis, because histologic evaluation may be compromised by profound sepsis and tissue putrefaction. Accuracy in diagnosis may have significant public health and medicolegal consequences.

Platelets and stroke.

Platelets are anucleate cells with little or no capacity for de novo protein synthesis. Their potential haemostatic reactivity is established at or before thrombopoiesis by their precursor cell, the bone marrow megakaryocyte. In some pathologic conditions, the megakaryocyte-platelet-haemostatic axis (MPHA) becomes perturbed, resulting in the formation of hyperfunctional platelets which may contribute to the development of vascular disease or an acute thrombotic event such as ischaemic stroke or myocardial infarction. Laboratory measurements of platelet function have established that platelet reactivity is accentuated in acute ischaemic stroke, particularly following cortical rather than lacunar infarction. Whether accentuated platelet function is a cause or a consequence of stroke is not yet clear, but it is likely that patients with certain risk factor profiles have some degree of platelet activation preceding the stroke. Further work into the MPHA is required to establish whether enhanced post-stroke platelet reactivity can be referred to the megakaryocyte. The antiplatelet agents tested to date are effective in secondary but not primary prevention of stroke. This probably reflects the diverse pathophysiology of stroke: accentuated platelet function is only likely to be a significant factor in cortical stroke.

Bilateral Wilms' tumor: a clinicopathologic review.

Bilateral Wilms' tumor occurs at a younger age than unilateral disease. While it generally has a good prognosis, it presents a therapeutic dilemma to balance curative surgical resection with preservation of renal tissue. A 15 year review of bilateral Wilms' tumors diagnosed at Princess Margaret Hospital was undertaken. Of 46 Wilms' tumor cases, eight were designated bilateral by diagnostic imaging (median age 1.1 years compared with 3.5 years for unilateral tumors). The surgical management entailed primary nephrectomy with contralateral biopsy in two patients, and bilateral biopsy and delayed resection in all remaining surviving patients (one patient died of perioperative complications). Seven patients had localized disease (stage I/II) and the six surviving patients received chemotherapy with vincristine and actinomycin; no patient received radiotherapy. All are alive and well (median follow-up 5.1 years). The remaining patient presented with pulmonary metastases and died of disease progression. Pathologic review revealed that four patients had truly bilateral disease demonstrable by histology, three had unilateral Wilms' tumor with contralateral nephrogenic rests, and in one patient the biopsies of the contralateral kidney showed neither tumor nor nephrogenic rests. In most cases pathological material was subject to external review. Follow-up demonstrates excellent renal function with compensatory hypertrophy in the remaining renal tissue. Conservative surgery and simple out-patient based, low toxicity chemotherapy is curative in most patients.

Cytogenetics of an atypical Spitz nevus metastatic to a single lymph node.

Clinicopathologic details of an atypical Spitz nevus are reported. A metastasis to a single ipsilateral lymph node is documented. Cytogenetic analysis of the metastatic lesion revealed a clonal chromosomal abnormality, add(6)(q12-13), previously noted in cases of malignant melanoma.

Atypical mycobacterial lymphadenitis in childhood--a clinicopathological study of 17 cases.

AIMS: To assess the clinical and pathological features of atypical mycobacterial lymphadenitis in childhood to define the salient clinical and histological features. METHODS: 17 cases were included on the basis of positive culture or demonstration of bacilli of appropriate morphology and staining characteristics. RESULTS: The mean age at diagnosis was 4.86 years. All children were systemically well, with clear chest x rays. Unilateral cervical lymphadenopathy was the commonest mode of presentation. Differential Mantoux testing played no part in diagnosis. Clinical diagnosis improved with awareness. Treatment varied with surgeons opting for excision and paediatricians adding six months antituberculous chemotherapy. Acid- and alcohol-fast bacilli were identified in nine cases. Bacterial cultures were conducted in 16 cases and were positive for atypical or nontuberculous mycobacteria in 14, the main organism being M avium-intracellulare complex (11 cases). Histologically, 12 cases had bright eosinophilic serpiginous necrosis with nuclear debris scattered throughout the necrotic foci. Langhans type giant cells featured in the majority of cases but infiltration by plasma cells and neutrophils was not consistent. CONCLUSIONS: Atypical mycobacterial lymphadenitis of childhood represents a rare but significant disease with characteristic clinical and histological features.