Cellular dosimetry of [177Lu]Lu-DOTA-[Tyr3]octreotate radionuclide therapy: the impact of modeling assumptions on the correlation with in vitro cytotoxicity

Background: Survival and linear-quadratic model fitting parameters implemented in treatment planning for targeted radionuclide therapy depend on accurate cellular dosimetry. Therefore, we have built a refined cellular dosimetry model for [177Lu]Lu-DOTA-[Tyr3]octreotate (177Lu-DOTATATE) in vitro experiments, accounting for specific cell morphologies and sub-cellular radioactivity distributions. Methods: Time activity curves were measured and modeled for medium, membrane-bound, and internalized activity fractions over 6 days. Clonogenic survival assays were performed at various added activities (0.1–2.5 MBq/ml). 3D microscopy images (stained for cytoplasm, nucleus, and Golgi) were used as reference for developing polygonal meshes (PM) in 3DsMax to accurately render the cellular and organelle geometry. Absorbed doses to the nucleus per decay (S values) were calculated for 3 cellular morphologies: spheres (MIRDcell), truncated cone-shaped constructive solid geometry (CSG within MCNP6.1), and realistic PM models, using Geant4-10.03. The geometrical set-up of the clonogenic survival assays was modeled, including dynamic changes in proliferation, proximity variations, and cell death. The absorbed dose to the nucleus by the radioactive source cell (self-dose) and surrounding source cells (cross-dose) was calculated applying the MIRD formalism. Finally, the correlation between absorbed dose and survival fraction was fitted using a linear dose-response curve (high α/β or fast sub-lethal damage repair half-life) for different assumptions, related to cellular sha

Adding blood to agitated saline significantly improves detection of right-to-left shunt by contrast-transcranial color-coded duplex sonography.

Contrast-transcranial Doppler and contrast-transcranial color-coded duplex sonography (c-TCCD) have been reported to have high sensitivity in detecting patent foramen ovale as compared with transesophageal echocardiography. An international consensus meeting (Jauss and Zanette 2000) recommended that the contrast agent for right-to left-shunt (RLS) detection using contrast-transcranial Doppler be prepared by mixing 9mL of isotonic saline solution and 1mL of air. The aim of our study was to determine whether adding blood to the contrast agent results in improved detection of RLS. We enrolled all consecutive patients admitted to our neurosonology laboratory for RLS diagnosis. For each patient, we performed c-TCCD both at rest and during the Valsalva maneuver using two different contrast agents: ANSs (1mL of air mixed with 9mL of normal saline) and ANSHBs (1mL of air mixed with 8mL of normal saline and 1mL of the patient's blood). To classify RLS, we used a four-level visual categorization: (i) no occurrence of micro-embolic signals; (ii)grade I, 1-10 signals; (iii) grade II, >10 signals but no curtain; grade III, curtain pattern. We included 80 patients, 33 men and 47 women. RLS was detected in 18.8% at rest and in 35% during the Valsalva maneuver using ANSs, and in 31.3% and in 46.3% using ANSHBs, respectively (. p<0.0001). There was a statistically significant increase in the number of micro-embolic signals with the use of ANSHBs. The use of blood mixed with saline solution and air as a c-TCCD contrast agent produced an increase in positive tests and a higher grade of RLS compared with normal saline and air alone, either with or without the Valsalva maneuver

DNA Sequences of Parachlamydia-like organisms in CSF of Patients with Neurological Disorders

Previously our findings have suggested a possible association between C. pneumoniae and Chlamydia-like organism brain infections as a cofactor in Multiple Sclerosis (MS) development (Contini et al, 2008). In particular, the DNA sequences found exhibited 83.8% of similarity with Neochlamydia sp. LTUNC08556 16s rRNA and 67.9% with Endosymbiont of Acanthamoeba sp. UWC22 16s rRNA. On the basis of these results, and considering that other Chlamydia-related bacteria, including Simkania negevensis, Waddlia chondrophila and the recently discovered bacterium Protochlamydia naegleriophila might also cause human infections, we wanted to deepen and study other patients with different neurological disorders other than MS. A total of 105 CSF samples were collected from MS patients (n. 20), other inflammatory neurological diseases (n. 49, OIND) and non-inflammatory neurological diseases (n. 36, NIND). DNA was extracted from fresh CSF samples and PCR was carried out with primers targeting the 16S rRNA gene according to Greub (2006). We detected 7 (6,6%) positive specimens: 2 MS (10%), 2 OIND (4,1%) with polyneuritis and 3 NIND (8.3%) with chronic ischemia, parestesia and cerebral ischemic vasculopathy. In all cases the positive amplified PCR products corresponded to 261 bp. These were purified, sequenced and compared (BLAST program, http://www.ncbi. nlm.nih.gov/BLAST) with other gene sequences to assess the homology or percentage of identity. In MS and OIND patients, BLAST anaysis revealed Protochlamydia naegleriophila strain KNic 16S ribosomal RNA gene and Endosymbiont of Acanthamoeba sp. UWE1 16S ribosomal RNA gene (rates of homology 92%-93% and 99%-79%, respectively). In NIND patients, Endosymbiont of Acanthamoeba sp, Protochlamydia naegleriophila strain KNic 16S and Parachlamydia-related symbiont UWE25 (rates of homology 91%, 83% 81%, respectively) were found. The exact role of Parachlamydiaceae in human diseases and MS is not well defined. So far, Protochlamydia naegleriophila, a new uncultivable intracellular bacterium, was recently detected as potential agent of community-acquired pneumonia but its role is yet under investigation. The detection of this pathogen in MS as well as in neurological disorders is a new finding which may have potential clinical implications but needs to be further explored

Chlamydia pneumoniae-specific intrathecal oligoclonal antibody response is predominantly detected in a subset of multiple sclerosis patients with progressive forms.

The purpose of this study was to verify the actual involvement of Chlamydia pneumoniae in multiple sclerosis (MS) by the evaluation of its specific intrathecal humoral immune response in MS. We measured by enzyme-linked immunosorbent assay (ELISA) technique cerebrospinal fluid (CSF) and serum levels of anti-C. pneumoniae immunoglobulin G (IgG) in 27 relapsing-remitting (RR), 9 secondary progressive (SP), and 5 primary progressive (PP) MS patients, grouped according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. Twenty-one patients with other inflammatory neurological disorders (OIND) and 21 with noninflammatory neurological disorders (NIND) were used as controls. Quantitative intrathecal synthesis of anti-C. pneumoniae IgG was determined by antibody-specific index (ASI), whereas the presence of C. pneumoniae-specific CSF oligoclonal IgG bands was assessed by antigen-specific immunoblotting. ASI values indicative of C. pneumoniae-specific intrathecal IgG synthesis were present in a small proportion of MS (29.3%), OIND (33.3%), and NIND (4.8%) patients and were significantly more frequent (P < .05) in total MS and in OIND than in NIND and in SP (P < .01) and PP MS (P < .05) than in RR MS. C. pneumoniae-specific CSF-restricted OCB were detected only in three SP, one PP, and one RR MS patients. These findings suggest that an intrathecal production of anti-C. pneumoniae IgG is part of humoral polyreactivity driven by MS chronic brain inflammation. However, an intrathecal release of C. pneumoniae-specific oligoclonal IgG can occur in a subset of patients with MS progressive forms in whom a C. pneumoniae-persistent brain infection may play a pathogenetic role

Detection of Chlamydia pneumoniae-specific intrathecal oligoclonal antibody response in a subset of patients with progressive forms of multiple sclerosis

Purpose: Multiple Sclerosis (MS) is a presumed autoimmune chronic inflammatory demyelinating disease of the Central Nervous System (CNS) of unknown etiology. Epidemiological studies suggest that MS pathogenesis could be related to an infection superimposed on a predisposing genetic background. Currently, a growing body of data suggests that C. pneumoniae could cause a brain chronic persistent infection acting as a cofactor in the development of the disease. As an intrathecal oligoclonal antibody response directed against the causative agent is usually present in CNS infections, the aim of this study was to verify the distribution C. pneumoniae-specific CSF-restricted oligoclonal IgG bands (OCB) in MS patients and controls. Materials and methods: We measured by ELISA technique cerebrospinal fluid (CSF) and serum levels of anti-C. pneumoniae IgG in 27 relapsing-remitting (RR), 9 secondary progressive (SP) and 5 primary progressive (PP) MS patients grouped according to clinical and Magnetic Resonance Imaging (MRI) evidence of disease activity. Twenty-one patients with other inflammatory neurological disorders (OIND) and 21 with non-inflammatory neurological disorders (NIND) served as neurological controls. To avoid confounding factors, patients with HIV encephalopathy, Alzheimer’s disease and cerebrovascular diseases were excluded from the study Quantitative intrathecal synthesis of anti-C. pneumoniae IgG was determined by Antibody Specific Index (ASI). In all patients with C. pneumoniae-specific intrathecally produced antibodies, the presence of C. pneumoniae-specific CSF OCB was assessed by affinity-mediated immunoblot (AMI). After checking data for normality, statistical analysis was performed by Mann-Whitney U and Chi-square tests (χ2). Bonferroni correction was used for multiple comparisons. Results: Detectable levels of CSF and serum anti-C. pneumoniae IgG were more frequent in total MS and in OIND than in NIND patients. Accordingly, CSF and serum anti-C. pneumoniae IgG mean levels were higher in total MS and in OIND than in NIND patients. However, no significant differences were found for these variables among the various groups. ASI values suggestive of C. pneumoniae-specific intrathecal IgG synthesis were present in a small proportion of MS (12/41; 29.3%), OIND (7/21; 33.3%) and NIND (1/21; 4.8%) patients and were significantly more represented (p < 0.05) in total MS and in OIND than in NIND and in SP (p < 0.01) and PP MS (p < 0.05) than in RR MS. No additional statistical differences were observed for CSF and serum anti-C. pneumoniae IgG mean concentrations as well as for ASI values among RR, SP and PP MS and between MS patients with and without clinical and MRI evidence of disease activity. Among the patients with intrathecally produced anti-C. pneumoniae IgG, C. pneumoniae-specific CSF-restricted OCB were detected only in 3 SP, 1 PP and 1 RR MS patients. Conclusions: These findings confirm that the presence of an intrathecal humoral immune response to C. pneumoniae is not selectively restricted to MS, but is shared by several inflammatory neurological conditions. In addition, our results suggest that an intrathecal production of C. pneumoniae-specific oligoclonal IgG can occur in a subset of patients with MS progressive forms in which a C. pneumoniae brain chronic persistent infection may play an important pathogenetic role

EBV-specific intrathecal oligoclonal antibody response in multiple sclerosis patients

Although Epstein-Barr virus (EBV) has recently received increasingly attention as a potential causative infectious agent in Multiple Sclerosis (MS), the significance of EBV-specific humoral immune response in MS still remains to be clarified. The aim of our study was to investigate CSF and serum levels and the presence of an intrathecal synthesis of anti-EBV IgG in MS and controls. We measured by ELISA technique cerebrospinal fluid (CSF) and serum levels of anti-EBV IgG in 100 relapsing-remitting (RR) MS patients, grouped according to clinical and Magnetic Resonance Imaging (MRI) evidence of disease activity, in 109 patients with other inflammatory neurological disorders (OIND) and in 87 patients with non-inflammatory neurological disorders (NIND). Anti-EBV nuclear antigen-1 (EBNA-1) and anti viral capsid antigen (VCA) IgG levels were expressed as arbitrary units and quantitative intrathecal synthesis of anti-EBNA-1 and anti-VCA IgG was determined by Antibody Index (AI). The presence of EBV-specific CSF oligoclonal IgG bands (OCB) was assessed in MS patients by antigen-specific immunoblotting. CSF concentrations were higher in OIND than in MS (p < 0.0001) and NIND (p < 0.01) for anti-VCA IgG, and in MS than in NIND (p < 0.01) and in OIND than in NIND (p < 0.05) for anti-EBNA-1 IgG. Serum levels of anti-EBNA-1 IgG were more elevated in in MS than in OIND and NIND (p < 0.0001). Serum titers of anti-EBNA-1 IgG were inversely (p < 0.001) correlated with EDSS. An intrathecal IgG production of anti-VCA and anti-EBNA-1 IgG, as indicated by Antibody Index, was present only in a small proportion of MS and controls (range from 1.1 to 6.4%). EBV-specific CSF-restricted OCB were detected in 25/100 (25%) MS patients. Serum levels of anti-VCA IgG were greater in MS patients without than in those with EBV-specific CSF OCB. These findings argue against a direct pathogenetic role of EBV-targeted humoral immune response in MS. However, an intrathecal release of EBV-specific oligoclonal IgG can occur in a subset of patients with MS in whom an EBV brain persistent infection may act as a cofactor in the development of the disease. Accordingly, serum anti-EBNA-1 IgG response seems to mark MS patients compared to other inflammatory and non inflammatory conditions. Work supported by FISM (2008-R-12) and by Programma di ricerca Regione-Università 2007-200