Prophylactic and therapeutic efficacy of nitazoxanide against Cryptosporidium parvum in experimentally challenged neonatal calves

Diarrhoea caused by Cryptosporidium parvum is a major problem in calves younger than 4 weeks of age. To date only a few compounds have been approved for prophylactic and none for therapeutic use. Nitazoxanide (NTZ) has proven its efficacy in vitro against C. parvum and is approved by FDA for the treatment of human cryptosporidiosis. In a first experimental study, 3 uninfected calves were treated with NTZ and pharmacokinetics was followed through blood samples. Serum samples of uninfected treated calves contained both NTZ metabolites (tizoxanide and tizoxanide glucuronide) and oral administration at 12 h intervals was considered as optimal. Three groups of three calves (1-3 days old) were then each inoculated with 1x10(7) oocysts of C. parvum (cattle genotype): the prophylactic group received 15 mg/kg body weight NTZ twice daily orally in milk from 1 day before to 8 days postinoculation (dpi). The therapeutic group received the same dosage of NTZ for 10 days from the appearance of diarrhoea (between 1 and 5 dpi). The control group was left untreated. All calves were monitored daily from day -1 to 28 dpi and faecal samples were collected for evaluation of consistency and for determination of oocyst numbers per gram (OPG) of faeces. Diarrhoea was observed in all calves within the first week. Neither prophylactic nor therapeutic use of NTZ improved the clinical appearance and calves of the therapeutic showed a longer diarrheic episode (p<0.05) with strong altered faecal consistency compared to the untreated control group. The number of days with oocyst excretion did not differ significantly between the groups. In 5 out of 6 infected and treated calves oocyst excretion stopped only after discontinuation of treatment. In the prophylactic and in the control group mean values of the sum of the daily OPG per calf (8.5x10(6) and 8.0x10(6), respectively) and of the mean daily number of OPG (0.3x10(6) and 0.3x10(6), respectively) were similar, while the therapeutic group showed significantly lower values (1.9x10(6) and 0.06x10(6), respectively, p<0.05). However oocyst determinations in this group may have been altered by the severe diarrhoea, diluting oocyst densities in the analysed faecal samples. In conclusion, these preliminary results about the first prophylactic and therapeutic use of NTZ in calves did not show the expected positive effect on the course of the Cryptosporidium-infection, neither on reducing the clinical severity, nor on oocyst excretion.

Targeting autophagy as a potential therapeutic approach for melanoma therapy

Melanoma, occurring as a rapidly progressive skin cancer, is resistant to current chemo- and radiotherapy, especially after metastases to distant organs has taken place. Most chemotherapeutic drugs exert their cytotoxic effect by inducing apoptosis, which, however, is often deficient in cancer cells. Thus, it is appropriate to attempt the targeting of alternative pathways, which regulate cellular viability. Recent studies of autophagy, a well-conserved cellular catabolic process, promise to improve the therapeutic outcome in melanoma patients. Although a dual role for autophagy in cancer therapy has been reported, both protecting against and promoting cell death, the potential for using autophagy in cancer therapy seems to be promising. Here, we review the recent literature on the role of autophagy in melanoma with respect to the expression of autophagic markers, the involvement of autophagy in chemo- and immunotherapy, as well as the role of autophagy in hypoxia and altered metabolic pathways employed for melanoma therapy.

A papain-induced disc degeneration model for the assessment of thermo-reversible hydrogel-cells therapeutic approach

Nucleus pulposus (NP) regeneration by the application of injectable cell-embedded hydrogels is an appealing approach for tissue engineering. We investigated a thermo-reversible hydrogel (TR-HG), based on a modified polysaccharide with a thermo-reversible polyamide [poly(N-isopropylacrylamide), pNIPAM], which is made to behave as a liquid at room temperature and hardens at > 32 °C. In order to test the hydrogel, a papain-induced bovine caudal disc degeneration model (PDDM), creating a cavity in the NP, was employed. Human mesenchymal stem cells (hMSCs) or autologous bovine NP cells (bNPCs) were seeded in TR-HG; hMSCs were additionally preconditioned with rhGDF-5 for 7 days. Then, TR-HG was reversed to a fluid and the cell suspension injected into the PDDM and kept under static loading for 7 days. Experimental design was: (D1) fresh disc control + PBS injection; (D2) PDDM + PBS injection; (D3) PDDM + TR-HG (material control); (D4) PDDM + TR-HG + bNPCs; (D5) PDDM + TR-HG + hMSCs. Magnetic resonance imaging performed before and after loading, on days 9 and 16, allowed imaging of the hydrogel-filled PDDM and assessment of disc height and volume changes. In gel-injected discs the NP region showed a major drop in volume and disc height during culture under static load. The RT–PCR results of injected hMSCs showed significant upregulation of ACAN, COL2A1, VCAN and SOX9 during culture in the disc cavity, whereas the gene expression profile of NP cells remained unchanged. The cell viability of injected cells (NPCs or hMSCs) was maintained at over 86% in 3D culture and dropped to ~72% after organ culture. Our results underline the need for load-bearing hydrogels that are also cyto-compatible.

An MEKC assay for the therapeutic drug monitoring of cefepime

The development of a robust assay based on MEKC for cefepime in human serum and plasma with internal quality assurance is reported. Sample preparation comprises protein precipitation in the presence of SDS at pH 4.5. This is a gentle approach for which decomposition of cefepime during sample handling is negligible. After hydrodynamic sample injection of the supernatant, analysis occurs in a phosphate/borate buffer at pH 9.1 with 75 mM SDS using normal polarity and analyte detection at 257 nm. The MEKC run time interval and throughput are about 5 min and seven samples per hour, respectively. The calibration range for cefepime is 1-60 μg/mL, with 1 μg/mL being the LOQ. The performance of the assay with multilevel internal calibration was assessed with calibration and control samples. The assay is shown to be simple, inexpensive, reproducible, and robust. It was applied to determine cefepime levels in the sera of critically ill patients and to assess the instability of cefepime in patient and control samples. Our data revealed that serum containing cefepime can be stored at -20°C for a short time, whereas for long-term storage, samples have to be kept at -70°C.

Multiplex Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Therapeutic Drug Monitoring of Ribavirin, Boceprevir, and Telaprevir

New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy.

Bullous pemphigoid: therapeutic algrorithm and practical management

Introduction: Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease of the skin and mucosae. BP typically affects the elderly and manifests with severe itch, localised or generalised eczematous, urticated and/or bullous lesions. Its morbidity and impact on the quality of life are important. The disease is significantly associated with neurological disorders, such as stroke, Parkinson disease, major cognitive impairment and multiple sclerosis. Diagnosis of BP critically relies on immunopathologic examinations, particularly direct immunofluorescence microscopy studies. Areas covered: This paper looks at the evidence of therapies commonly used in bullous pemphigoid. Expert opinion: Treatment of BP has been a challenge, given the relative rarity of the disease, lack of good quality randomised controlled trials, the presence of co-morbidities in the affected elderly population and the high mortality rate. Recent controlled studies have indicated that potent topical corticosteroids constitute a more effective therapy for BP when compared to oral corticosteroids in terms of control of the disease, side effect profile and overall survival. Other therapies have been employed with varying success, but are not validated yet. Improved knowledge of the pathophysiology of BP will hopefully allow the development of new immunomodulatory treatments for this debilitating disease.