Enhancing Sensitivity of Detection of Immune Responses to Mycobacterium leprae Peptides in Whole-Blood Assays

Although worldwide leprosy prevalence has been reduced considerably following multidrug therapy, new case detection rates remain relatively stable, suggesting that transmission of infection still continues. This calls for new efforts, among which is development of assays that can identify subclinical/early-stage Mycobacterium leprae-infected subjects, a likely source of transmission. Areas in which leprosy is endemic often lack sophisticated laboratories, necessitating development of field-friendly immunodiagnostic tests for leprosy, like short-term whole-blood assays (WBA). In classical, peripheral blood mononuclear cell (PBMC)-based gamma interferon (IFN-γ) release assays, M. leprae peptides have been shown to discriminate in a more specific fashion than M. leprae proteins between M. leprae-exposed contacts and patients as opposed to healthy controls from the same area of endemicity. However, peptides induced significantly lower levels of IFN-γ than did proteins, particularly when whole blood was used. Therefore, possibilities of specifically enhancing IFN-γ production in response to M. leprae peptides in 24-h WBA were sought by addition of various cytokines and antibodies or by mannosylation of peptides. In addition, other cytokines and chemokines were analyzed as potential biomarkers in WBA. We found that only interleukin 12 (IL-12), not other costimulants, increased IFN-γ production in WBA while maintaining M. leprae peptide specificity, as evidenced by lack of increase of IFN-γ in control samples stimulated with IL-12 alone. The IL-12-induced increase in IFN-γ was mainly mediated by CD4⁺ T cells that did not produce IL-2 or tumor necrosis factor (TNF). Mannosylation further allowed the use of 100-fold-less peptide. Although not statistically significantly, macrophage inflammatory protein 1β (MIP-1β) and macrophage c protein 1 (MCP-1) levels specific for M. leprae peptide tended to be increased by IL-12. IP-10 production was also found to be a useful marker of M. leprae peptide responses, but its production was enhanced by IL-12 nonspecifically. We conclude that IFN-γ-based WBA combined with IL-12 represents a more sensitive and robust assay for measuring reactivity to M. leprae peptides.Leprosy is a disabling and stigmatizing disease caused by infection with Mycobacterium leprae. The characteristic immunological and clinical leprosy spectrum, classified by Ridley and Jopling in 1966 (25), ranges from tuberculoid (TT) or paucibacillary (PB) leprosy to lepromatous (LL) or multibacillary (MB) leprosy. In between these poles the borderline states borderline lepromatous (BL), borderline borderline (BB), and borderline tuberculoid (BT) leprosy are positioned. TT/BT patients in general show high cellular responses to M. leprae antigens injected in the skin as well as in in vitro T-cell assays; have low antibody titers to M. leprae antigens, including phenolic glycolipid I (PGL-I); and develop localized granulomatous disease with few, if any, detectable bacilli in their lesions. At the opposite end of the spectrum are LL/BL patients with a characteristic inability to generate M. leprae-specific Th1-cell responses and with disseminating progressive infection and high antibody titers to M. leprae antigens, including the M. leprae-specific cell surface antigen PGL-I.Over the last 2 decades the WHO leprosy elimination program, partly in combination with wide coverage of Mycobacterium bovis BCG vaccination (28), has had a massive effect on the registered number of cases, which dropped from approximately 5.4 million in 1985 to 212,802 worldwide at the beginning of 2008. In addition, since 2003 the global number of new cases detected showed a drastic decrease at an average rate of nearly 20% per year, and a reported year-end prevalence below 1 per 10,000 was obtained in 2007 in all countries with a population of >1 million except for Brazil, Nepal, and East Timor (32). However, part of the decrease was achieved by changing leprosy control policies and does not necessarily reflect the reality of infection. Concomitantly, the elimination campaign has had a severe downside as it led to a discontinuation of leprosy control programs and a decrease in leprosy clinics, specialists, and research. Thus, leprosy patients have to be treated in integrated programs, where health workers lack the knowledge and time to diagnose and treat leprosy. This resulted in sustained transmission as evidenced by the hundreds of thousands of new cases of leprosy that keep being detected globally every year (254,525 in 2007) and a 3.1% increase between 2007 and 2008 of new case detection in children (32). In addition, countries that do not exceed this prevalence rate nationwide still harbor regions of high endemicity, where leprosy remains a public health problem (e.g., Angola, Central African Republic, India, and Tanzania). These figures demonstrate that M. leprae-infected contacts and persons with subclinical, undiagnosed leprosy, likely the major sources of unidentified transmission, are an incessant source of active transmission, making early detection of leprosy or M. leprae infection and prompt multidrug treatment (MDT) of utmost importance for control of the disabling effects of leprosy.Diagnosis of leprosy is at present based only on clinical features and the number of skin lesions. Due to the loss of diagnostic skills and the decrease in skin smear services, the detection of M. leprae infection occurs in many cases only after significant and irreversible nerve damage has occurred. Since M. leprae is not cultivable in vitro, bacterial enumeration by microscopic examination is required for leprosy classification, monitoring chemotherapy regimens, and diagnosis of relapse, yielding, in inexperienced hands, data of limited specificity and sensitivity. There is no test available that can detect asymptomatic M. leprae infection or predict progression of infection to clinical disease. Assays that demonstrate the presence of IgM antibodies against PGL-I are useful for most MB patients but have limited value in identifying or predicting PB patients who typically develop cellular rather than humoral immunity (23).In order to assess host immune responses after exposure to or infection with mycobacteria, the ex vivo whole-blood assay (WBA) is a helpful test. In the past, several variations of the WBA have been introduced in which unseparated heparinized blood is stimulated with antigen either overnight or for as long as 6 days, after which plasma or supernatant is analyzed for cytokines (8). Since WBAs are much simpler and faster than conventional assays using peripheral blood mononuclear cells (PBMC), they have been successfully applied in commercially available gamma interferon (IFN-γ) release assays (IGRAs) for diagnosis of tuberculosis (TB) (QuantiFERON-TB-Gold-In-Tube [QFT-G-IT]). These IGRAs have a number of advantages over skin tests (e.g., Mitsuda or Mantoux tests), as they can measure directly from whole blood within a single day, do not require repeated visits for reading of the test, and do not carry the inherent risk of boosting upon repeated testing. Furthermore, there are no exclusion criteria, as the test can also be performed for patients with skin disorders.To measure responses to recombinant M. leprae proteins and whole-cell sonicate of M. leprae, 6-day WBAs measuring IFN-γ in diluted blood samples have proved successful at field sites in various countries such as Nepal (33) and Malawi (4, 34). Since 6-day WBAs require access to a CO₂ incubator, our investigations focused on the development of a practical diagnostic assay on 24-h WBA. Furthermore, as (mixtures of) M. leprae peptides proved to induce more specific IFN-γ release by PBMC than did recombinant M. leprae proteins (16, 29), we previously used M. leprae peptides for the 24-h WBA (17) in analogy to the successful peptide-based QuantiFERON-TB-Gold-In-Tube (11). However, the shortened incubation period hampered the use of M. leprae peptides in WBA, as IFN-γ production induced was weak in comparison to that when M. leprae proteins were used (17, 29). Therefore, in the present study we analyzed other cytokines/chemokines as potential readout for WBA and tested possible enhancement of IFN-γ production in response to M. leprae peptides by addition of several cytokines, antibodies against factors that block the optimal release of IFN-γ, or antibodies that can trigger costimulation.     

Clinical effects of transcatheter hepatic arterial embolization with holmium-166 poly(l-lactic acid) microspheres in healthy pigs

PURPOSE: The aim of this study is to evaluate the toxicity of holmium-166 poly(L: -lactic acid) microspheres administered into the hepatic artery in pigs. METHODS: Healthy pigs (20-30 kg) were injected into the hepatic artery with holmium-165-loaded microspheres ((165)HoMS; n = 5) or with holmium-166-loaded microspheres ((166)HoMS; n = 13). The microspheres' biodistribution was assessed by single-photon emission computed tomography and/or MRI. The animals were monitored clinically, biochemically, and ((166)HoMS group only) hematologically over a period of 1 month ((165)HoMS group) or over 1 or 2 months ((166)HoMS group). Finally, a pathological examination was undertaken. RESULTS: After microsphere administration, some animals exhibited a slightly diminished level of consciousness and a dip in appetite, both of which were transient. Four lethal adverse events occurred in the (166)HoMS group due either to incorrect administration or comorbidity: inadvertent delivery of microspheres into the gastric wall (n = 2), preexisting gastric ulceration (n = 1), and endocarditis (n = 1). AST levels were transitorily elevated post-(166)HoMS administration. In the other blood parameters, no abnormalities were observed. Nuclear scans were acquired from all animals from the (166)HoMS group, and MRI scans were performed if available. In pigs from the (166)HoMS group, atrophy of one or more liver lobes was frequently observed. The actual radioactivity distribution was assessed through ex vivo (166m)Ho measurements. CONCLUSION: It can be concluded that the toxicity profile of HoMS is low. In pigs, hepatic arterial embolization with (166)HoMS in amounts corresponding with liver-absorbed doses of over 100 Gy, if correctly administered, is not associated with clinically relevant side effects. This result offers a good perspective for upcoming patient trials.     

186Re-etidronate in breast cancer patients with metastatic bone pain.

The aim of this study was to evaluate the efficacy of 186Re-1,1-hydroxyethylidene diphosphonate (etidronate) in breast cancer patients with painful bone metastases. METHODS: Thirty patients with advanced breast cancer who had metastatic bone pain were treated with 186Re-etidronate using different dosages in a noncomparative, open-label study. Twenty-four patients were evaluated for efficacy (6 patients had incomplete datasets). Dosages varied from 1295 to 2960 MBq (35 to 80 mCi). Efficacy was evaluated according to the multidimensional pain model using a paper-and-pencil diary. The diary was kept twice daily for 8-10 wk (2 wk before through 6-8 wk after 186Re-etidronate treatment). Response was determined with a strict criteria, in which pain intensity (PI), medication index (MI) and daily activities (DA) were core determinants. Response was defined as: (a) Reduced PI > or = 5% while MI and DA were at least constant; or (b) Reduced PI <25% in combination with improvement of MI or DA > or = 25%, without worsening of either factor. Duration of response should always exceed a minimum of 2 wk. RESULTS: Fifty-eight percent (n = 14) of all patients reported a response. The maximum follow-up period was 8 wk. Duration of response ranged from 2 to 8 wk (mean 4 wk). Patients (14/24) not only experienced considerable pain reduction, but in 12 patients this was also accompanied by noteworthy reduction in MI (> or = 25%). No clear dose-response relationship was found. CONCLUSION: With strict pain assessment criteria, 186Re-etidronate showed a response of 58% in the palliative treatment of metastatic bone pain originating from breast cancer.     

Long-term toxicity of holmium-loaded poly(L-lactic acid) microspheres in rats

The aim of this study was to get insight into the toxic effects of holmium-166-loaded poly(L-lactic acid) microspheres (Ho-PLLA-MS) which have very interesting features for treatment of liver malignancies. Acute, mid- and long-term effects were studied in healthy Wistar rats by evaluating clinical, biochemical and tissue response. Rats were divided into four treatment groups: sham, decayed neutron-irradiated Ho-PLLA-MS, non-irradiated Ho-PLLA-MS and PLLA-MS. After implantation of the microspheres into the liver of the rats, the animals were monitored (body weight, temperature and liver enzymes) for a period of 14-18 months. Some of the rats that received previously neutron-irradiated Ho-PLLA-MS were periodically scanned with magnetic resonance imaging (MRI) to see if holmium was released from the microspheres. After sacrifice, the liver tissue was histologically evaluated. Bone tissue was subjected to neutron-activation analysis in order to examine whether accumulation of released holmium in the bone had occurred. No measurable clinical and biochemical toxic effects were observed in any of the treatment groups. Furthermore, histological analyses of liver tissue samples only showed signs of a slight chronic inflammation and no significant differences in the tissue reaction between rats of the different treatment groups could be observed. The non-irradiated PLLA-MS and Ho-PLLA-MS stayed intact during the study. In contrast, 14 months after administration, the neutron-irradiated Ho-PLLA-MS was not completely spherical anymore, indicating that degradation had started. However, the holmium loading had not been released as was illustrated with MRI and affirmed by neutron-activation analysis of bone tissue. In conclusion, neutron-irradiated Ho-PLLA-MS does not provoke any toxic reaction and can be applied safely in vivo.     

Hybrid scatter correction applied to quantitative holmium-166 SPECT

Ho-166 is a combined beta-gamma emitter of which the betas can be used therapeutically. From the 81 keV gammas of Ho-166, SPECT images can be obtained, which give opportunities to guide Ho-166 therapy. Accurate reconstruction of Ho-166 images is currently hampered by photopeak-scatter in the patient, down-scatter in the detector, collimator and patient caused by the 1.4 MeV photons and by bremsstrahlung. We developed and validated a method for quantitative SPECT of Ho-166 that involves correction for both types of scatter plus non-uniform attenuation correction using attenuation maps. Photopeak-scatter (S) is compensated for by a rapid 3D Monte Carlo (MC) method that is incorporated in ordered subset (OS) reconstruction of the emission data, together with simultaneous correction for attenuation (A) and detector response (D); this method is referred to as OS-ADS. Additionally, for correction of down-scatter, we use a 14 keV wide energy window centred at 118 keV (OS-ADSS). Due to a limited number of available energy windows, the same 118 keV energy window was used for down-scatter correction of the simultaneously acquired Gd-153 transmission data. Validations were performed using physical phantom experiments carried out on a dual-head SPECT system; Gd-153 transmission line sources were used for acquiring attenuation maps. For quantitative comparison of OS-ADS and OS-ADSS, bottles filled with Ho-166 were placed in both a cylindrical phantom and an anthropomorphic thorax phantom. Both OS-ADS and OS-ADSS were compared with an ordered subset reconstruction without any scatter correction (OS-AD). Underestimations of about 20% in the attenuation map were reduced to a few per cent after down-scatter correction. The average deviation from the true activity contained in the bottles was +72% with OS-AD. Using OS-ADS, this average overestimation was reduced to +28% and with OS-ADSS the deviation was further reduced to 16%. With OS-AD and OS-ADS, these numbers were more sensitive to the choice of volumes of interest than with OS-ADSS. For the reconstructed activity distributions, erroneous background activity found with OS-AD was reduced by a factor of approximately 2 by applying OS-ADS and reduced by a factor of approximately 4 by applying OS-ADSS. The combined attenuation, photopeak-scatter and down-scatter correction framework proposed here greatly enhanced the quantitative accuracy of Ho-166 imaging, which is of the uppermost importance for image-guided therapies. It is expected that the method, with adapted window settings, also can be applied to other isotopes with high energy peaks that contaminate the photopeak data, such as I-131 or In-111.     

Quantification of in vivo spiperone binding in the rat striatum after lesions produced by kainate or decortication

The potential of in vivo spiperone binding as a tool for the detection and quantitative analysis of striatal dopamine (DA) receptor alterations was studied in rat brain lesioned in several ways. Two weeks after kainate (KA) injection a significantly higher radioactivity accumulation was observed in the lesioned striatum than in the contralateral structure after a tracer dose of [3H]spiperone. The difference was maximal 2 days after surgery and it was present for at least 4 weeks while it was reversed 11 weeks after KA injection. The radioactivity uptake (tracer dose of [3H]spiperone) measured 2 weeks after surgery could be specifically prevented in both KA-lesioned and contralateral striatum by haloperidol and N-n-propylnorapomorphine while non-dopaminergic drugs were almost without effect. More than 80% of the radioactivity accumulation was saturable in both contralateral (unlesioned) and KA-lesioned striatum, leaving a slightly higher non-saturable radioactivity level in the latter. One week after unilateral ablation of the cerebral cortex overlying the striatum only minor bilateral differences in striatal radioactivity content were found after a tracer dose of [3H]spiperone. No differences were present after 6-OHDA lesion of the nigrostriatal pathway. Striatal DA receptor densities (Bmax) were determined from the dose-dependency of total striatal spiperone accumulation. This relationship was assessed using cerebellar spiperone accumulation instead of dose. Thus a Bmax of about 75 fmol X mg-1 tissue was found in the striatum of control (unoperated) rats and contralateral to the striatal KA lesion while 2 weeks after surgery it was approximately 33 fmol X mg-1 in the KA-lesioned striatum. One week after unilateral decortication Bmax values of about 50 and 65 fmol X mg-1 were found ipsi- and contralaterally to the lesion respectively.     

Removal of chloroform from biodegradable therapeutic microspheres by radiolysis

Radioactive holmium-166 loaded poly(l-lactic acid) microspheres are promising systems for the treatment of liver malignancies. These microspheres are loaded with holmium acetylacetonate (HoAcAc) and prepared by a solvent evaporation method using chloroform. After preparation the microspheres (Ho-PLLA-MS) are activated by neutron irradiation in a nuclear reactor. It was observed that relatively large amounts of residual chloroform (1000-6000 ppm) remained in the microspheres before neutron irradiation. Since it is known that chloroform is susceptible for high-energy radiation, we investigated whether neutron and gamma irradiation could result in the removal of residual chloroform in HoAcAc-loaded and placebo PLLA-MS by radiolysis. To investigate this, microspheres with relatively high and low amounts of residual chloroform were subjected to irradiation. The effect of irradiation on the residual chloroform levels as well as other microsphere characteristics (morphology, size, crystallinity, molecular weight of PLLA and degradation products) were evaluated. No chloroform in the microspheres could be detected after neutron irradiation. This was also seen for gamma irradiation at a dose of 200 kGy phosgene, which can be formed as the result of radiolysis of chloroform, was not detected with gas chromatography-mass spectrometry (GC-MS). A precipitation titration showed that radiolysis of chloroform resulted in the formation of chloride. Gel permeation chromatography and differential scanning calorimetry showed a decrease in molecular weight of PLLA and crystallinity, respectively. However, no differences were observed between irradiated microsphere samples with high and low initial amounts of chloroform. In conclusion, this study demonstrates that neutron and gamma irradiation results in the removal of residual chloroform in PLLA-microspheres.     

Hepatic aldehyde dehydrogenase (E.C. 1.2.1.3.) isoenzymes and malondialdehyde levels in alcoholic liver disease

To clarify the controversially discussed behaviour of hepatic ALDH isozymes in different liver diseases, 98 liver biopsy specimens were investigated as to their total ALDH and their isozyme activity. Simultaneously the malondialdehyde (MDA) content was measured in both biopsy samples and serum. The results show a significant decrease of the "low Km" isozyme E-2 activity depending on the severity of the liver disease. The tendency for the E-2 activity to decrease was greater in alcoholic than in nonalcoholic liver diseases. Although in all our diagnosis groups elevated MDA concentrations could be measured in both serum and liver tissue, a direct specific inhibitory effect on the E-2 activity could not be confirmed. The change from the low Km pathway to the high Km pathway of aldehyde detoxification, however, may contribute to the progress in alcoholic liver disease.     

Holmium Nanoparticles: Preparation and <Emphasis Type="Italic">In Vitro</Emphasis> Characterization of a New Device for Radioablation of Solid Malignancies

Purpose  

The present study introduces the preparation and in vitro characterization of a nanoparticle device comprising holmium acetylacetonate for radioablation of unresectable solid malignancies.     

Targeting of liver tumour in rats by selective delivery of holmium-166 loaded microspheres: a biodistribution study.

Intra-arterial administration of beta-emitting particles that become trapped in the vascular bed of a tumour and remain there while delivering high doses, represents a unique approach in the treatment of both primary and metastatic liver tumours. Studies on selective internal radiation therapy of colorectal liver metastases using yttrium-90 glass microspheres have shown encouraging results. This study describes the biodistribution of 40-microm poly lactic acid microspheres loaded with radioactive holmium-166, after intra-arterial administration into the hepatic artery of rats with implanted liver tumours. Radioactivity measurements showed >95% retention of injected activity in the liver and its resident tumour. The average activity detected in other tissues was < or =0.1%ID/g, with incidental exceptions in the lungs and stomach. Very little 166Ho activity was detected in kidneys (<0.1%ID/g), thereby indicating the stability of the microspheres in vivo. Tumour targeting was very effective, with a mean tumour to liver ratio of 6. 1+/-2.9 for rats with tumour (n=15) versus 0.7+/-0.5 for control rats (n=6; P<0.001). These ratios were not significantly affected by the use of adrenaline. Histological analysis showed that five times as many large (>10) and medium-sized (4-9) clusters of microspheres were present within tumour and peritumoural tissue, compared with normal liver. Single microspheres were equally dispersed throughout the tumour, as well as normal liver parenchyma.