Comparison of the depot effect and immunogenicity of liposomes based on dimethyldioctadecylammonium (DDA), 3β-[N-(N',N'-Dimethylaminoethane)carbomyl] cholesterol (DC-Chol), and 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP): prolonged liposome retention mediates stronger Th1 responses

The immunostimulatory capacities of cationic liposomes are well-documented and are attributed both to inherent immunogenicity of the cationic lipid and more physical capacities such as the formation of antigen depots and antigen delivery. Very few studies have however been conducted comparing the immunostimulatory capacities of different cationic lipids. In the present study we therefore chose to investigate three of the most well-known cationic liposome-forming lipids as potential adjuvants for protein subunit vaccines. The ability of 3β-[N-(N',N'-dimethylaminoethane)carbomyl] cholesterol (DC-Chol), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), and dimethyldioctadecylammonium (DDA) liposomes incorporating immunomodulating trehalose dibehenate (TDB) to form an antigen depot at the site of injection (SOI) and to induce immunological recall responses against coadministered tuberculosis vaccine antigen Ag85B-ESAT-6 are reported. Furthermore, physical characterization of the liposomes is presented. Our results suggest that liposome composition plays an important role in vaccine retention at the SOI and the ability to enable the immune system to induce a vaccine specific recall response. While all three cationic liposomes facilitated increased antigen presentation by antigen presenting cells, the monocyte infiltration to the SOI and the production of IFN-γ upon antigen recall was markedly higher for DDA and DC-Chol based liposomes which exhibited a longer retention profile at the SOI. A long-term retention and slow release of liposome and vaccine antigen from the injection site hence appears to favor a stronger Th1 immune response.     

The vesicle size of DDA:TDB liposomal adjuvants plays a role in the cell-mediated immune response but has no significant effect on antibody production

The use of cationic liposomes as experimental adjuvants for subunit peptide of protein vaccines is well documented. Recently the cationic liposome CAF01, composed of dimethyldioctadecylammonium (DDA) and trehalose dibehenate (TDB), has entered Phase I clinical trials for use in a tuberculosis (TB) vaccine. CAF01 liposomes are a heterogeneous population with a mean vesicle size of 500 nm; a strong retention of antigen at the injection site and a Th1-biassed immune response are noted. The purpose of this study was to investigate whether CAF01 liposomes of significantly different vesicle sizes exhibited altered pharmacokinetics in vivo and cellular uptake with activation in vitro. Furthermore, the immune response against the TB antigen Ag85B-ESAT-6 was followed when various sized CAF01 liposomes were used as vaccine adjuvants. The results showed no differences in vaccine (liposome or antigen) draining from the injection site, however, significant differences in the movement of liposomes to the popliteal lymph node were noted. Liposome uptake by THP-1 vitamin D3 stimulated macrophage-like cells did not show a liposome size-dependent pattern of uptake. Finally, whilst there were no significant differences in the IgG1/2 regardless of the liposome size used as a delivery vehicle for Ag85B-ESAT-6, vesicle size has a size dependent effect on cell proliferation and IL-10 production with larger liposomes (in excess of 2 μm) promoting the highest proliferation and lowest IL-10 responses, yet vesicles of ~ 500 nm promoting higher IFN-γ cytokine production from splenocytes and higher IL-1β at the site of injection.     

Whole-body MRI for the detection of bone marrow involvement in lymphoma: prospective study in 116 patients and comparison with FDG-PET

Objective

To assess and compare the value of whole-body MRI with FDG-PET for detecting bone marrow involvement in lymphoma.

Methods

A total of 116 patients with newly diagnosed lymphoma prospectively underwent whole-body MRI and blind bone marrow biopsy (BMB) of the posterior iliac crest. Of 116 patients, 80 also underwent FDG-PET. Patient-based sensitivities of whole-body MRI for detecting bone marrow involvement were calculated using BMB as reference standard and compared with FDG-PET in aggressive and indolent lymphomas separately.

Results

Sensitivity of whole-body MRI in all lymphomas was 45.5 % [95 % confidence interval (CI): 29.8–62.0 %]. Sensitivity of whole-body MRI in aggressive lymphoma [88.9 % (95 % CI: 54.3–100 %)] was significantly higher (P?=?0.0029) than that in indolent lymphoma [23.5 % (95 % CI: 9.1–47.8 %)]. Sensitivity of FDG-PET in aggressive lymphoma [83.3 % (95 % CI: 41.8–98.9 %)] was also significantly higher (P?=?0.026) than that in indolent lymphoma [12.5 % (95 % CI: 0–49.2 %)]. There were no significant differences in sensitivity between whole-body MRI and FDG-PET (P?=?1.00)

Conclusion

Sensitivity of whole-body MRI for detecting lymphomatous bone marrow involvement is too low to (partially) replace BMB. Sensitivity of whole-body MRI is significantly higher in aggressive lymphoma than in indolent lymphoma and is equal to FDG-PET in both entities.

Key Points

? Bone marrow involvement in lymphoma has prognostic and therapeutic implications. ? Blind bone marrow biopsy (BMB) is standard for bone marrow assessment. ? Neither whole-body MRI nor FDG-PET can yet replace BMB. ? Both techniques have higher sensitivity in aggressive than in indolent lymphoma. ? Both imaging techniques are complementary to BMB.     

Safety and efficacy of deep brain stimulation in refractory cluster headache: a randomized placebo-controlled double-blind trial followed by a 1-year open extension

Chronic cluster headache (CCH) is a disabling primary headache, considering the severity and frequency of pain attacks. Deep brain stimulation (DBS) has been used to treat severe refractory CCH, but assessment of its efficacy has been limited to open studies. We performed a prospective crossover, double-blind, multicenter study assessing the efficacy and safety of unilateral hypothalamic DBS in 11 patients with severe refractory CCH. The randomized phase compared active and sham stimulation during 1-month periods, and was followed by a 1-year open phase. The severity of CCH was assessed by the weekly attacks frequency (primary outcome), pain intensity, sumatriptan injections, emotional impact (HAD) and quality of life (SF12). Tolerance was assessed by active surveillance of behavior, homeostatic and hormonal functions. During the randomized phase, no significant change in primary and secondary outcome measures was observed between active and sham stimulation. At the end of the open phase, 6/11 responded to the chronic stimulation (weekly frequency of attacks decrease >50%), including three pain-free patients. There were three serious adverse events, including subcutaneous infection, transient loss of consciousness and micturition syncopes. No significant change in hormonal functions or electrolytic balance was observed. Randomized phase findings of this study did not support the efficacy of DBS in refractory CCH, but open phase findings suggested long-term efficacy in more than 50% patients, confirming previous data, without high morbidity. Discrepancy between these findings justifies additional controlled studies (clinicaltrials.gov number NCT00662935).     

Healing time, long-term result and effects of stem cell treatment in acute tympanic membrane perforation

OBJECTIVE: The incidence of otitis media in children between the age of 2 and 6 years is well documented. Repeated attacks may cause acute and chronic perforations. The surgical treatment for repairing chronic perforation is quite uncomfortable for the patients of this age group because of the invasiveness of this treatment. The aim of this study was to determine the long-term influence of embryonic stem cells on acute perforations and the effect of gelatin as a vehicle for applied stem cells. The possibility of teratogenic effects of the stem cells was also observed. METHODS: Bilateral laser myringotomy was performed in 17 adult Sprague-Dawley rats, divided into two groups. Gelatin, a substance suitable as vehicle for bioactive material was used bilaterally around the perforation in group A, to serve as a scaffold for repairing tissue. The stem cells were used in the right tympanic membrane perforation leaving the left tympanic membrane as a control. The animals in group B received the same treatment except for the use of gelatin and in addition received an immuno-suppressive agent. After half a year of observation the mechanical stiffness of the tympanic membrane was measured by moiré interferometry for group B and the morphological study was performed by light microscopy for both groups A and B and electron microscopy for group A. RESULTS: Stem cell treated ears did not show any enhanced healing of the perforation although a marked thickening of the lamina propria was observed compared with control group. After half a year the strength and the stiffness of the tympanic membrane was almost the same for both treated and untreated ears. No evidence of teratoma was found after half a year. CONCLUSION: This study suggests that the stem cells stimulate the proliferation of connective tissue and fibers in the lamina propria, possibly mediated by secreted substances, although the stiffness properties do not seem to be altered. The use of gelatin does not seem to enhance the healing process of the tympanic membrane perforation.     

Fresh tympanic membrane perforations heal without significant loss of strength.

HYPOTHESIS: The mechanical and structural properties of the tympanic membrane change after a perforation has healed. BACKGROUND: In previous studies, efforts have been made to enhance the healing process of tympanic membrane perforations. The strength of the healed perforation has been tested with moiré interferometry in gerbils, but in no other species. METHODS: A laser myringotomy was made on 10 Sprague-Dawley rats and 10 CBA mice, and assessments were made after 2 or 4 weeks with moiré interferometry and light and electron microscopy. RESULTS: The mean peak displacement at pressure loads of +350 daPa and of -350 daPa did not differ significantly in the healed perforations as compared with the untouched tympanic membranes. Morphologic assays showed fivefold increased thickness at the site of the perforation due to invaded fibroblasts and extracellular matrix. CONCLUSION: Moiré interferometry was successfully performed in the rat ears, whereas in mouse ears the method was not easily applicable due to technical difficulties. The stress-strain curve of the rat tympanic membrane displays an S-shape. The strength of the spontaneously healed tympanic membrane after myringotomy was not significantly impaired. The site of the perforation became significantly thickened at 2 and 4 weeks post-myringotomy. This information is of clinical importance, because recently closed perforations will be challenged by pressure gradient in everyday life.