Immunocryosurgery for basal cell carcinoma: results of a pilot, prospective, open-label study of cryosurgery during continued imiquimod application

Background/aim  Theoretical considerations support the combination of cryosurgery and topical imiquimod to treat basal cell carcinomas (BCC). The aim of the present study was to test the feasibility and efficacy of 'cryosurgery during continued imiquimod application' ('immunocryosurgery') to treat 'high-risk-for-recurrence' BCCs.
Methods  Thirteen patients with 21 biopsy-proven tumours (4 of 21 relapses after prior surgery) were included. After 2–5 weeks (median, 3) of daily 5% imiquimod cream application, the tumours were treated by liquid N₂ cryosurgery (spray, two cycles, 10–20 s) and imiquimod was continued for additional 2–12 weeks (median, 4). The outcome after at least 18 months of follow-up (18–24 months) is currently reported.
Results  Nineteen of 21 tumours responded promptly to immunocryosurgery; two tumours required additional treatment cycles to clear. Thus, the clinical clearance rate was 100%. Only 1 of 21(5%) tumour relapsed after at least 18 months of follow-up (cumulative efficacy: 95%).
Conclusions  'Immunocryosurgery' is a promising non-surgical combination modality to treat 'high-risk-for-recurrence BCCs'. Initial evidence is suggestive of an at least additive effect of the two combined modalities. Further studies comparing immunocryosurgery directly with cryosurgery and imiquimod monotherapies will confirm the reported results.     

FEASIBILITY OF CONDUCTING CARDIOVASCULAR OUTCOME RESEARCH IN AUSTRALIAN GENERAL PRACTICE: RESULTS FROM THE ANBP2 PILOT STUDY

1. The present study aimed to determine the feasibility of conducting a 5 year cardiovascular outcome trial of the treatment of 6000 elderly hypertensive patients in Australian general practices. 2. General practitioners (GPs) were invited to participate by mail and personal follow-up. Patient records were reviewed to identify subjects for a blood pressure (BP) screening programme. Blood pressure was measured on three occasions and eligible subjects were included if the average BP was 160 mmHg systolic or 90 mmHg diastolic if systolic BP was 140 mmHg. 3. Seven hundred and forty-one GPs were approached and 89 were enrolled in the study (12% of mail invites and 75% of those receiving a personal contact). In 16 practices where screening was completed, 82 000 records were reviewed to identify 4% patients eligible for screening. Twenty-two per cent of eligible subjects attended screening. Of 1938 subjects screened, 180 (9%) had BP 5=160/90 mmHg. Forty-seven percent of subjects (n = 916) were receiving antihypertensive therapy and 184 (20%) were withdrawn from therapy. One hundred and sixteen (63%) of these subjects had BP return to study entry levels within 6 weeks. Fifty-seven newly diagnosed and 81 previously treated subjects were randomized (7% of the screened population). 4. Based on the high participation rate of GPs, the response rate of patients to attend a BP screening programme and the 7% randomization to screening ratio for entry into the study, the ANBP2 pilot study has demonstrated that it is feasible to recruit subjects from Australian general practices to a cardiovascular outcome trial.     

GGA1 acts as a spatial switch altering amyloid precursor protein trafficking and processing

The beta-amyloid (Abeta) precursor protein (APP) is cleaved sequentially by beta-site of APP-cleaving enzyme (BACE) and gamma-secretase to release the Abeta peptides that accumulate in plaques in Alzheimer's disease (AD). GGA1, a member of the Golgi-localized gamma-ear-containing ARF-binding (GGA) protein family, interacts with BACE and influences its subcellular distribution. We now report that overexpression of GGA1 in cells increased the APP C-terminal fragment resulting from beta-cleavage but surprisingly reduced Abeta. GGA1 confined APP to the Golgi, in which fluorescence resonance energy transfer analyses suggest that the proteins come into close proximity. GGA1 blunted only APP but not notch intracellular domain release. These results suggest that GGA1 prevented APP beta-cleavage products from becoming substrates for gamma-secretase. Direct binding of GGA1 to BACE was not required for these effects, but the integrity of the GAT (GGA1 and TOM) domain of GGA1 was. GGA1 may act as a specific spatial switch influencing APP trafficking and processing, so that APP-GGA1 interactions may have pathophysiological relevance in AD.     

Development of an MRI contrast agent for both detection and inhibition of the amyloid-β fibrillation process

A curcumin derivative conjugated with Gd-DO3A (Gd-DO3A-Comp.B) was synthesised as an MRI contrast agent for detecting the amyloid-β (Aβ) fibrillation process. Gd-DO3A-Comp.B inhibited Aβ aggregation significantly and detected the fibril growth at 20 μM of Aβ with 10 μM of probe concentration by T₁-weighted MR imaging.

A curcumin derivative conjugated with Gd-DO3A (Gd-DO3A-Comp.B) was developed to significantly inhibit the amyloid-β (Aβ) aggregation and detect the fibril growth by T₁-weighted MR imaging.

A significant increase of Alzheimer''s disease (AD) patients urges the development of therapeutic and diagnostic technology.¹ As with the therapeutic development, diagnostic technology also faces several obstacles. To date, the definite diagnosis of AD relies on the histopathological data of post-mortem.^2,3 The non-invasive imaging technology targeting AD biomarkers such as amyloid β (Aβ) could provide phenotypical diagnostics, although the development of Aβ probes still remains challenging. Several contrast agents for single photon emission computed tomography (SPECT) and positron emission tomography (PET) such as Florbetapir-¹⁸F and Pittsburgh compound-B ([¹¹C]PiB) were developed as efficient tracers in mild cognitive impairment patients.^4,5 However, PET- and SPECT-based diagnostics require injection of radioactive probes, which cannot be measured frequently due to radiation exposure and limited availability of facilities. They also provide limited information on the anatomic profile of biomarkers due to their low spatial resolution and imprecise microscopic localization.⁶ In contrast, magnetic resonance imaging (MRI) contrast agents could quantify the Aβ accumulation in the anatomic brain image.⁷Several reported MRI contrast agents using gadolinium (Gd) complexes demonstrate potential use of Aβ detection. A clinically approved contrast agent, Gd(iii) diethylenetriaminepentaacetic acid (Gd-DTPA) complex accumulates in brain after opening the blood–brain barrier (BBB) by using mannitol and detects Aβ deposits in the mice AD-model.⁸ To improve the selectivity, Gd complexes were conjugated with compounds binding to Aβ such as Pittsburgh compound B (Gd-DO3A-PiB) which also serves as an approach for increasing MRI sensitivity.^9,10 An α,β-unsaturated ketone compound curcumin has been widely reported as an Aβ probe due to its ability to bind the hydrophobic site of Aβ.^11,12 Allen et al. firstly reported the direct conjugation of curcumin with Gd-DTPA which binds to Aβ with four times higher relaxivity than free Gd-DTPA.¹³ Furthermore, a polymalic acid-based nanoparticle covalently linked with curcumin and Gd-DOTA could also detect Aβ in human brain specimen by MRI.¹⁴ These previous studies demonstrate that the curcumin structure has significant potential for the development of MRI contrast agents for AD diagnosis.Previously, we reported a curcumin derivative, compound B, possesses 100-times stronger inhibitory activity of Aβ aggregation than curcumin on the basis of thioflavin T (ThT) competitive binding assay.^15,16 According to this result, we designed curcumin-based Gd probes for the detection and inhibition of Aβ (Fig. 1A–C). We hypothesized that these probes could accelerate proton longitudinal relaxation depending on the fibrillation stage of Aβ, because molecular tumbling rate of the Gd complexes becomes slower (Fig. 1A).¹⁷ As a result, the probes permit the detection of Aβ by longitudinal relaxation time (T₁)-weighted imaging. This mechanism could also be utilized to estimate the inhibitory activity of the probes by T₁-based analysis (Fig. 1B). The curcumin and compound B were directly conjugated with the macrocyclic DO3A ligand through the propylamine linker to obtain Gd-DO3A-Cur and Gd-DO3A-Comp.B, respectively (Fig. 1C).Open in a separate windowFig. 1(A) A probe concept that produces T₁ in a dependent manner of Aβ fibrillation process. (B) Inhibitor-based probes that cause moderate T₁ decreases due to inhibitory activity of fibrillation. (C) The chemical structures of the synthesized Gd probes for Aβ detection and inhibition.Gd-DO3A-Cur and Gd-DO3A-Comp.B were synthesized according to Scheme 1 (detail in Scheme S1, ESI^†). The compound 5a and 5b, which have asymmetric curcumin derivatives containing carboxylic acid group, were synthesized by three step reactions. Amide bond formation with DO3A(tBu)₃-propylamine ligand¹⁸ by condensation reaction afforded compound 7a and 7b. The tert-butyl groups were deprotected by trifluoroacetic acid producing compound 8a and 8b. The complexation was performed with GdCl₃·6H₂O by adjusting the reaction pH to 7, giving 43 and 41% yields of Gd-DO3A-Cur and Gd-DO3A-Comp.B, respectively. The T₁ relaxivities (r₁) of the curcumin-based Gd probes were estimated by T₁ measurement using a 1 tesla NMR relaxometry (Fig. S1, ESI^†). For the comparison, we synthesized Gd-DO3A-Chal which is a reported probe for Aβ.¹⁹ The r₁ of Gd-DO3A-Comp.B, Gd-DO3A-Cur, and Gd-DO3A-Chal were 7.1, 6.1 and 5.3 mM⁻¹ s⁻¹, respectively. These r₁ values are higher than that of clinically approved Gd-DOTA (3.9 mM⁻¹ s⁻¹).²⁰ The molecular weight of Gd-DO3A-Comp.B and Gd-DO3A-Cur is almost two times larger than that of Gd-DOTA. Because the r₁ increases approximately linearly with molecular weight in low magnetic field,¹⁷ the high r₁ values of Gd-DO3A-Comp.B and Gd-DO3A-Cur might be mainly attributed to their high rotational correlation time, rather than the high number of coordinated water molecules. The r₁ of Gd-DO3A-Chal was comparable to the value reported previously.¹⁹Open in a separate windowScheme 1Synthetic scheme of Gd-DO3A-Cur and Gd-DO3A-Comp.B. (a) B(OH)₃, morpholine, DMF, 100 °C, 10 min. (b) 3a/3b, B(OH)₃, morpholine, DMF, 100 °C, 10 min. (c) TFA, DCM. (d) DO3A(tBu)₃-propylamine ligand, PyBOP, HOBt, Et₃N, DMF. (e) 7a/7b, TFA, DCM. (f) GdCl₃·6H₂O, NaOH, H₂O.We evaluated the inhibitory effect of three probes toward Aβ aggregation by Congo red assay.²¹ After 24 h incubation of 20 μM Aβ with 10 μM probe, Gd-DO3A-Comp.B showed the lowest fluorescence intensity, indicating the strongest inhibitory activity followed by Gd-DO3A-Cur (Fig. 2A). As the comparison, the reported MRI agents, Gd-DO3A-Chal showed slight inhibitory activity. The inhibitory effect was further evaluated by transmission electron microscopy (TEM) with negative staining (Fig. 2B). In the absence of the probes, Aβ formed huge and massive fibril similar to the typical morphology of Aβ fibril.²² The TEM images of Aβ with Gd-DO3A-Comp.B showed the presence of white spheres below 10 nm, demonstrating that Gd-DO3A-Comp.B strongly inhibits Aβ aggregation. In fact, the fibril growth stopped at a stage of oligomer formation. Lower inhibitory activity of Gd-DO3A-Cur was also found to provide a shortened worm-like fibril, which is the typical morphology of Aβ exposed to curcumin.²³ In contrast, the small amount of white spheres and partial fibril disruption were found in the image of Aβ with Gd-DO3A-Chal. In comparison with a reported Gd-DTPA-curcumin possessing inhibitory activity starting at 50 μM, Gd-DO3A-Comp.B possessed stronger inhibition of Aβ aggregation at 10 μM.²⁴ The MTT assay using Neuro 2a cells showed that IC₅₀ of Gd-DO3A-Cur and Gd-DO3A-Comp.B. were more than 500 μM, indicating that these compounds did not possess significant cytotoxicity (Fig. S2, ESI^†).Open in a separate windowFig. 2Inhibitory effect of the Gd probes toward Aβ aggregation measured by Congo red assay (A) and negative staining TEM images (B). The Gd probes were co-incubated with monomeric Aβ for 24 h in PBS at pH 7.4. [Gd] = 10 μM, [Aβ] = 20 μM. Scale bars = 100 nm.To detect fibrillation process by NMR relaxometry, we measured T₁ of the probe mixture with Aβ which were pre-incubated for 1, 3, 6, 12, and 24 h to make it form the fibrils of different growth stages (Fig. 3A and B). The T₁ of Gd-DO3A-Comp.B solution decreased with pre-incubation time of Aβ, demonstrating that the Gd-DO3A-Comp.B can detect Aβ fibril depending on the growth stage (Fig. 3B). Lower T₁ involved with Aβ growth could be caused by the reduction in tumbling rate of the Gd complex.²⁵ We also co-incubated the probes with the Aβ monomer and monitored T₁ changes over the incubation time (Fig. 3A, B and S3, ESI^†). Interestingly, the Gd-DO3A-Comp.B did not cause significant T₁ decreases even after 24 hours co-incubation with Aβ monomers, demonstrating that Gd-DO3A-Comp.B has a strong inhibitory effect on fibril formation and the inhibition can be monitored by T₁ measurement (Fig. 3B). The inhibitory effect was consistent with the results of Congo red assay and TEM (Fig. 2). On the other hand, the time-dependent increases of T₁ were observed in Gd-DO3A-Chal and Gd-DO3A-Cur. This might be because these two probes were buried in the hydrophobic pocket as Aβ fibril grew up and fewer water molecules permitted access to the Gd ions. It is also possible that these probes have lower binding affinity, especially for matured fibril, and require higher concentrations to produce significant T₁ changes.²⁶ These probe did not produce the significant ΔT₁ between monomer and fibril samples (Fig. 3B and S3, ESI^†), although they showed little inhibition in Congo red assay and TEM (Fig. 2).Open in a separate windowFig. 3(A) Experimental design of T₁-based detection of Aβ fibrillation and inhibition by using the Gd probes. (B) T₁ changes of the Gd probe solutions with pre-incubated fibrils and monomers in PBS at pH 7.4 (mean ± SEM, n = 3). [Gd] = 10 μM, [Aβ] = 20 μM.The feasibility of the Gd probes was further evaluated by in vitro MRI measurement using a 1 tesla scanner. The T₁-weighted images showed that Gd-DO3A-Comp.B produced slight T₁ signal increases with Aβ monomers for 2 and 24 h (Fig. 4A and B). More significant signal increases were observed in the Gd-DO3A-Comp.B with Aβ fibril pre-incubated for 24 h (Fig. 4C). In contrast, Gd-DO3A-Chal and Gd-DO3A-Cur did not show significant signal changes in the presence of Aβ monomers or fibrils (Fig. 4A–C). These results were mostly consistent with the T₁ profile measured by NMR (Fig. 3). Compared to the previously reported Gd-DO3A-Chal that required 100 μM of the probe concentration to detect the equimolar Aβ,¹⁹ Gd-DO3A-Comp.B could detect five-times lower concentration of Aβ (20 μM) with ten-times lower probe concentration (10 μM). Therefore, Gd-DO3A-Comp.B could be promising to further develop highly sensitive diagnostic MRI contrast agents of AD.Open in a separate windowFig. 4 T ₁-weighted images of the Gd probe solutions in the presence of monomeric Aβ at 2 h incubation (A), monomeric Aβ at 24 h incubation (B), and Aβ fibrils pre-incubated for 24 h (C). Incubation was conducted in PBS at pH 7.4.In conclusion, we synthesized the curcumin-based Gd probes which enabled the detection and inhibition of Aβ fibril formation. Gd-DO3A-Comp.B allowed for the highly sensitive detection of Aβ fibril by the T₁ measurement. Moreover, the inhibitory activity could be estimated by T₁ measurement, because Gd-DO3A-Comp.B decreased T₁ depending on the growth stage of Aβ fibril formation. Such unique modality would be useful not only for the diagnostics but also for the direct evaluation of the therapeutic efficacy in vivo. For the future application, it would be important to combine with BBB penetration methods targeting the brain such as transient opening of the BBB using focused ultrasound or mannitol injection.^27,28     

Mechanism of spinal cord injury regeneration and the effect of human neural stem cells-secretome treatment in rat model

BACKGROUND Globally, complete neurological recovery of spinal cord injury(SCI) is still less than 1%, and 90% experience permanent disability. The key issue is that a pharmacological neuroprotective-neuroregenerative agent and SCI regeneration mechanism have not been found. The secretomes of stem cell are an emerging neurotrophic agent, but the effect of human neural stem cells(HNSCs) secretome on SCI is still unclear.AIM To investigate the regeneration mechanism of SCI and neuroprotective-neuro...     

Genome Profiling of SARS-CoV-2 in Indonesia,ASEAN and the Neighbouring East Asian Countries: Features,Challenges and Achievements

Whole-genome sequencing (WGS) has played a significant role in understanding the epidemiology and biology of SARS-CoV-2 virus. Here, we investigate the use of SARS-CoV-2 WGS in Southeast and East Asian countries as a genomic surveillance during the COVID-19 pandemic. Nottingham–Indonesia Collaboration for Clinical Research and Training (NICCRAT) initiative has facilitated collaboration between the University of Nottingham and a team in the Research Center for Biotechnology, National Research and Innovation Agency (BRIN), to carry out a small number of SARS-CoV-2 WGS in Indonesia using Oxford Nanopore Technology (ONT). Analyses of SARS- CoV-2 genomes deposited on GISAID reveal the importance of clinical and demographic metadata collection and the importance of open access and data sharing. Lineage and phylogenetic analyses of two periods defined by the Delta variant outbreak reveal that: (1) B.1.466.2 variants were the most predominant in Indonesia before the Delta variant outbreak, having a unique spike gene mutation N439K at more than 98% frequency, (2) Delta variants AY.23 sub-lineage took over after June 2021, and (3) the highest rate of virus transmissions between Indonesia and other countries was through interactions with Singapore and Japan, two neighbouring countries with a high degree of access and travels to and from Indonesia.     

Neurocysticercosis in radiographically imaged seizure patients in U.S. emergency departments

Neurocysticercosis appears to be on the rise in the United States, based on immigration patterns and published cases series, including reports of domestic acquisition. We used a collaborative network of U.S. emergency departments to characterize the epidemiology of neurocysticercosis in seizure patients. Data were collected prospectively at 11 university-affiliated, geographically diverse, urban U.S. emergency departments from July 1996 to September 1998. Patients with a seizure who underwent neuroimaging were included. Of the 1,801 patients enrolled in the study, 38 (2.1%) had seizures attributable to neurocysticercosis. The disease was detected in 9 of the 11 sites and was associated with Hispanic ethnicity, immigrant status, and exposure to areas where neurocysticercosis is endemic. This disease appears to be widely distributed and highly prevalent in certain populations (e.g., Hispanic patients) and areas (e.g., Southwest).