Clinical, metabolic, and molecular genetic characterization of hereditary methemoglobinemia caused by cytochrome b5 reductase deficiency in 30 dogs.

Genotype-phenotype correlations of humans and dogs with hereditary methemoglobinemia are not yet well characterized. We determined total hemoglobin and methemoglobin (MetHb) concentrations, cytochrome b5 reductase (CYB5R) enzyme activities, genotypes, and clinical signs in 30 dogs with persistent cyanosis without cardiopulmonary disease. Erythrocytic CYB5R enzyme activities were low in all dogs assayed. Owner-reported quality of life ranged from subclinical to occasional exertional syncope. Two previously reported and two novel CYB5R3 missense variants were identified among the methemoglobinemic cohort and were predicted to impair enzyme function. Two variants were recurrent: a homozygous Ile194Leu substitution was found in Pomeranians and other small dogs, and a homozygous Arg219Pro change occurred predominately in pit bull terriers. The other two variants were Thr202Ala and Gly76Ser substitutions in single dogs. Of the two common CYB5R3 genotypes, Arg219Pro was associated with a more severe metabolic phenotype. We conclude that CYB5R3 deficiency is the predominate cause of canine hereditary methemoglobinemia. Although this finding is unlikely to alter the clinical approach to hereditary methemoglobinemia in dogs, it demonstrates the possibility of how genotype-phenotype cohort analysis might facilitate precision medicine in the future in veterinary medicine.

A homozygous ADAMTS2 nonsense mutation in a Doberman Pinscher dog with Ehlers Danlos syndrome and extreme skin fragility.

An eight-week old Doberman Pinscher was diagnosed with Ehlers Danlos syndrome based on the dog's hyper-mobile carpal, tarsal and stifle joints and abnormal skin. The skin was loose and hyper-elastic with several wounds and large atrophic scars. The dog was euthanized after a severe degloving injury from minimal trauma. A whole-genome sequence, generated with DNA from the dog's blood, contained a rare, homozygous C-to-T transition at position 2408978 on chromosome 11. This transition is predicted to alter the ADAMTS2 transcript (ADAMTS2:c.769C>T) and encode a nonsense mutation (p.Arg257Ter). Biallelic ADAMTS2 mutations have caused a type of Ehlers Danlos syndrome known as dermatosparaxis in other species.

Tomosynthesis in musculoskeletal pathology.

Tomosynthesis is an imaging technique that uses standard X-ray equipment with digital flat panel detectors to create tomographic images from very low-dose projections obtained at different angles. These images are parallel to the plane of the detector. Filtered back-projection or iterative reconstruction algorithms can be used to produce them. Iterative reconstruction used with a metal artifact reduction algorithm reduces metal artifacts, and therefore, improve image quality and in-depth spatial resolution. The radiation dose is lower compared to that of computed tomography and is two to three times the dose of a standard radiography. Tomosynthesis is intended for the analysis of high-contrast structures and especially for bones. It is superior to projection radiography when bone superimpositions are important or when metal structures hide regions of interest. The high in-plane resolution and its ability to perform exams in weight-bearing positioning are some of the main advantages of this technique. The impossible production of perpendicular multiplanar reconstruction and a limited contrast resolution are its main limitations. Tomosynthesis must be considered as an extension or an addition to standard radiography, as it can be performed in the same diagnostic step. The purpose of this article was to describe the principles, advantages and limitations, and current and future applications in musculoskeletal pathology of tomosynthesis.

Long-term Treatment with Methylene Blue in a Dog with Hereditary Methemoglobinemia Caused by Cytochrome b5 Reductase Deficiency.

A juvenile male mixed breed dog was presented for lethargy, exercise intolerance, and aggression when touched on the head. Cyanosis, tachycardia, and tachypnea were observed and persisted during oxygen supplementation. Arterial blood gas analysis by co-oximetry identified an increased methemoglobin concentration (27%; normal, <2%) with normal arterial oxygen tension. The methemoglobinemia and associated clinical signs resolved after administration of methylene blue (1 mg/kg) IV, and the dog was discharged. The affected dog's whole-genome sequence contained 2 potentially causal heterozygous CYB5R3 missense mutations suggesting that cytochrome b5 reductase deficiency was responsible for the methemoglobinemia. This hypothesis was confirmed by enzyme analysis that identified cytochrome b5 reductase activity in the affected dog's erythrocytes to only approximately 6% of that in a control sample. Clinical signs recurred 11 days after discharge but normalized and the methemoglobin concentration decreased with methylene blue administration PO (1.5 mg/kg, initially daily and then every other day).

GM2 Gangliosidosis in Shiba Inu Dogs with an In-Frame Deletion in HEXB.

Consistent with a tentative diagnosis of neuronal ceroid lipofuscinosis (NCL), autofluorescent cytoplasmic storage bodies were found in neurons from the brains of 2 related Shiba Inu dogs with a young-adult onset, progressive neurodegenerative disease. Unexpectedly, no potentially causal NCL-related variants were identified in a whole-genome sequence generated with DNA from 1 of the affected dogs. Instead, the whole-genome sequence contained a homozygous 3 base pair (bp) deletion in a coding region of HEXB. The other affected dog also was homozygous for this 3-bp deletion. Mutations in the human HEXB ortholog cause Sandhoff disease, a type of GM2 gangliosidosis. Thin-layer chromatography confirmed that GM2 ganglioside had accumulated in an affected Shiba Inu brain. Enzymatic analysis confirmed that the GM2 gangliosidosis resulted from a deficiency in the HEXB encoded protein and not from a deficiency in products from HEXA or GM2A, which are known alternative causes of GM2 gangliosidosis. We conclude that the homozygous 3-bp deletion in HEXB is the likely cause of the Shiba Inu neurodegenerative disease and that whole-genome sequencing can lead to the early identification of potentially disease-causing DNA variants thereby refocusing subsequent diagnostic analyses toward confirming or refuting candidate variant causality.

[Dosimetric comparaison between VMAT and tomotherapy with para-aortic irradiation for cervix carcinoma]. / Comparaison entre arcthérapie volumétrique modulée et tomothérapie pour le cancer du col utérin, avec irradiation lomboaortique.

PURPOSE: Intensity-modulated radiotherapy (IMRT) has proven its dosimetric superiority over conformational radiotherapy for cervix cancers in terms of digestive toxicity. Volumetric modulated arctherapy (VMAT) has shown its dosimetric interest when compared to IMRT with static beams. The purpose of our study was to compare conformational radiotherapy, VMAT and tomotherapy for cervical cancers with para-aortic lymph nodes irradiation. PATIENTS AND METHODS: The dosimetric data from ten patients were compared between the three techniques, with collection of the dose received by the planning target volume, kidneys, bowel, rectum, bladder, bone marrow and spinal cord, as well as the complete dose. RESULTS: There was a significant difference in favour of VMAT and tomotherapy when compared with conformational radiotherapy for the organs at risk: intestines (V(20 Gy), V(30 Gy) and V(45 Gy)), rectum (V(30 Gy) and V(45 Gy)), bladder (V(30 Gy) and V(45 Gy)) and kidneys (V(12 Gy), Daverage, V(20 Gy)). Volumes receiving 20, 30 and 45 Gy were significantly higher in conformational radiotherapy than in VMAT and tomotherapy. There was a significant difference in favour of tomotherapy when compared with VMAT for V(12 Gy) in kidneys and V(45 Gy) in the bladder. CONCLUSION: This study confirms the interest of VMAT and tomotherapy for pelvic and para-aortic lymph nodes irradiation of cervix cancer when compared with conformational radiotherapy. There was little difference between VMAT and tomotherapy, except for kidney sparing, which was better with tomotherapy and thus may be interesting for patients receiving nephrotoxic chemotherapy.

[Radionuclides, technologies and quality control in brachytherapy]. / Radioéléments, appareillages et contrôle de qualité en curiethérapie.

Brachytherapy consists in placing radioactive sources directly in contact with the tumoral bed. The type of source, its characteristics and its use will be adapted regarding the kind of treatment foreseen. Modern brachytherapy techniques employ remote afterloading technologies allowing better quality and security in the delivery of treatment. This development of technology goes with an increase need of quality control of equipments, including radionuclides, and procedures.

CT dose optimisation and reduction in osteoarticular disease.

With an improvement in the temporal and spatial resolution, computed tomography (CT) is indicated in the evaluation of a great many osteoarticular diseases. New exploration techniques such as the dynamic CT and CT bone perfusion also provide new indications. However, CT is still an irradiating imaging technique and dose optimisation and reduction remains primordial. In this paper, the authors first present the typical doses delivered during CT in osteoarticular disease. They then discuss the different ways to optimise and reduce these doses by distinguishing the behavioural factors from the technical factors. Among the latter, the optimisation of the milliamps and kilovoltage is indispensable and should be adapted to the type of exploration and the morphotype of each individual. These technical factors also benefit from recent technological evolutions with the distribution of iterative reconstructions. In this way, the dose may be divided by two and provide an image of equal quality. With these dose optimisation and reduction techniques, it is now possible, while maintaining an excellent quality of the image, to obtain low-dose or even very low-dose acquisitions with a dose sometimes similar that of a standard X-ray assessment. Nevertheless, although these technical factors provide a major reduction in the dose delivered, behavioural factors, such as compliance with the indications, remain fundamental. Finally, the authors describe how to optimise and reduce the dose with specific applications in musculoskeletal imaging such as the dynamic CT, CT bone perfusion and dual energy CT.

[Comparison of irradiation for tomosynthesis and CT of the wrist]. / Comparaison de l'irradiation en scanner et tomosynthèse du poignet.

PURPOSE: Tomosynthesis is a novel imaging modality based on the use of flat-panel detector technology. Its impact in the evaluation of the post-traumatic wrist has yet to be defined. The low associated radiation dose is one of its main advantages. Comparison of radiation doses between tomosynthesis, radiographs and CT is thus necessary. MATERIALS AND METHODS: The delivered doses for a five-view wrist radiographic examination, frontal and lateral tomosynthesis and CT were measured using radiothermoluminescent dosimeters. RESULTS: Biplane tomosynthesis has the lowest delivered dose with 25% less radiation than a standard five-view radiographic examination (0.72 versus 0.96 mGy), and a 28-fold radiation reduction compared to CT (19.8 mGy). CONCLUSION: Tomosynthesis delivers less radiation than a standard radiographic examination. For the wrist, it results in a 28-fold reduction in radiation compared to CT.

[Statistical process control applied to intensity modulated radiotherapy pretreatment controls with portal dosimetry]. / Maîtrise statistique des processus appliquée aux contrôles avant traitement par dosimétrie portale en radiothérapie conformationnelle avec modulation d'intensité

PURPOSE: The first purpose of this study was to illustrate the contribution of statistical process control for a better security in intensity modulated radiotherapy (IMRT) treatments. This improvement is possible by controlling the dose delivery process, characterized by pretreatment quality control results. So, it is necessary to put under control portal dosimetry measurements (currently, the ionisation chamber measurements were already monitored by statistical process control thanks to statistical process control tools). The second objective was to state whether it is possible to substitute ionisation chamber with portal dosimetry in order to optimize time devoted to pretreatment quality control. PATIENTS AND METHODS: At Alexis-Vautrin center, pretreatment quality controls in IMRT for prostate and head and neck treatments were performed for each beam of each patient. These controls were made with an ionisation chamber, which is the reference detector for the absolute dose measurement, and with portal dosimetry for the verification of dose distribution. Statistical process control is a statistical analysis method, coming from industry, used to control and improve the studied process quality. It uses graphic tools as control maps to follow-up process, warning the operator in case of failure, and quantitative tools to evaluate the process toward its ability to respect guidelines: this is the capability study. The study was performed on 450 head and neck beams and on 100 prostate beams. RESULTS: Control charts, showing drifts, both slow and weak, and also both strong and fast, of mean and standard deviation have been established and have shown special cause introduced (manual shift of the leaf gap of the multileaf collimator). Correlation between dose measured at one point, given with the EPID and the ionisation chamber has been evaluated at more than 97% and disagreement cases between the two measurements were identified. CONCLUSION: The study allowed to demonstrate the feasibility to reduce the time devoted to pretreatment controls, by substituting the ionisation chamber's measurements with those performed with EPID, and also that a statistical process control monitoring of data brought security guarantee.

Inhibition of replication of HIV in primary monocyte/macrophages by different antiviral drugs and comparative efficacy in lymphocytes.

Several anti-HIV drugs acting on different steps of virus replication were tested in our experimental model of primary monocyte/macrophages; the results were compared with the activity found in lymphocytes. Nucleoside analogues (AZT, ddI, ddC, d4T, PMEA, 3TC etc.) show greater activity in macrophages (M/M) than in lymphocytes. In particular, the EC50 of AZT, ddC, and ddI in M/M is 2- to 100-fold lower than that found in lymphocytes. This greater efficacy of nucleoside analogues in M/M depends on the enhancement of their chain-terminating activity by the low levels of endogenous deoxynucleoside-triphosphates (dNTP) usually found in resting cells such as M/M. Non-nucleoside reverse transcriptase inhibitors (NNRTI) do not act as chain terminators (thus their antiviral effect is not related to the intracellular concentrations of dNTP); as a consequence the activity of TSAO, HEPT, TIBO, and other NNRTI tested in M/M is similar to that found in lymphocytes. Regarding inhibitors of binding and fusion of HIV, we found that their anti-HIV activity is markedly decreased (or even nullified) when M/M are treated with cytokine activators of M/M function and enhancers of HIV replication. More relevant from a clinical standpoint, protease inhibitors are able to inhibit HIV replication in chronically infected macrophages (i.e., cells carrying the proviral genome already integrated in the host genome). All other inhibitors of late stage of virus life cycle tested (antisense-rev, anti-tat, interferon-alpha and -gamma, phosphorothioate analogues, GLQ-223, etc.) were totally inactive in chronically infected macrophages. The different effects of various classes of HIV inhibitors in lymphocytes and macrophages suggests that AIDS therapy should consider all aspects of the pathogenesis of HIV infection and must be restricted to drugs, or combinations of drugs, active against both lymphocytes and M/M in all body compartments where the virus hides and replicates.

9-(2-Phosphonylmethoxyethyl) adenine increases the survival of influenza virus-infected mice by an enhancement of the immune system.

PMEA (9-(2-phosphonylmethoxyethyl)adenine) is a potent inhibitor of DNA viruses and retroviruses able to enhance natural immune functions such as natural killer cell activity and interferon production. The results reported in this paper show that the treatment with PMEA significatively decreased the mortality of mice challenged with influenza A/PR8 virus (an RNA virus, non sensitive to the antiviral effect of PMEA) compared to untreated, infected controls (median survival 8.64 days and 7.61 days, respectively), and reduced lung weight and consolidation (two surrogate markers of virus infection). Furthermore, virus titer obtained from lung homogenates was substantially decreased in PMEA-treated mice compared to controls. Finally, enhancement of natural killer cell activity was achieved in PMEA-treated A/PR8-infected mice compared to A/PR8-infected controls. Overall, results suggest that PMEA decreases the influenza virus-related mortality and morbidity through the enhancement of some immune functions, and that this effect might be additive or even synergystic with the direct inhibitory effect of DNA viruses or retroviruses induced by PMEA itself. This supports the importance of evaluating this drug in patients with diseases related to herpesviruses or to human immunodeficiency virus.

In vitro activity of inhibitors of late stages of the replication of HIV in chronically infected macrophages.

Because of the importance of macrophages in the pathogenesis of the disease caused by HIV, we investigated the efficacy of various anti-HIV drugs in human primary macrophages acutely or chronically infected by this virus. The results obtained for acutely infected macrophages show that dideoxynucleosides (AZT, ddI, and ddC), interferon-alpha and -gamma, mismatched double-stranded RNA, Tat inhibitor, phosphorothioate antisense, and inhibitors of HIV protease, all significantly inhibit virus replication at concentrations far below those toxic for the cells. However, in macrophages in which proviral DNA is already integrated (chronically infected macrophages), only the three inhibitors of HIV protease induced significant virus inhibition at concentrations 100 or more times higher than those effective in acutely infected macrophages. Treatment of macrophages with macrophage colony-stimulating factor does not affect the anti-HIV efficacy of protease inhibitors. These results suggest that therapeutic strategies with activity for macrophages, including inhibitors of HIV protease, are worth pursuing in patients with HIV infection.

Enhancement of natural killer activity and interferon induction by different acyclic nucleoside phosphonates.

Acyclic nucleoside phosphonate (ANP) analogues are a class of compounds with potent activity against herpesviruses and/or retroviruses. Our preliminary experiments have shown that 9-(2-phosphonylmethoxyethyl)adenine (PMEA), a prototype of the ANP family, enhances some parameters of natural immunity. In this paper we have evaluated the effect of different schedules of administration of PMEA and other ANP analogues of clinical interest upon natural killer (NK) activity and interferon (IFN) production in a mouse model. The results show that PMEA significantly enhances NK activity and interferon production. Other ANP analogues tested in our system, i.e., 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP), and 9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA), similarly induced enhancement of natural immunity. The immunomodulating effect of PMEA was even more pronounced with a single administration compared to repeated administrations of the drug. Dose-dependent enhancement of NK activity and IFN production could also be demonstrated during chronic administration of PMEA (more resembling to what will be the schedule of administration of this drug in patients). Overall, the data here presented suggest that the enhancement of some natural immune functions induced by ANP analogues may add to the direct antiviral activity of these drugs against retroviruses and herpesviruses, and thus may be able to increase the host resistance against viral infections.

Immunomodulatory activity of 9-(2-phosphonylmethoxyethyl)adenine (PMEA), a potent anti-HIV nucleotide analogue, on in vivo murine models.

In order to evaluate the influence of antiviral nucleoside analogues upon the natural immune system, we investigated the immunomodulatory activity of 9-(2-phosphonylmethoxyethyl)adenine (PMEA), a nucleotide analogue with potent anti-HIV and anti-herpes activity, in a murine system. C57BL/6 mice were inoculated intraperitoneally with 10, 25 and 50 mg PMEA/kg. Mononuclear cells were isolated from their spleens, and some natural immune functions were evaluated. The results show that PMEA significantly increases the levels of natural killer (NK)-cell cytotoxicity. We also found that alpha/beta IFN production was substantially increased in PMEA-treated mice, while both IL-1 and IL-2 production was decreased. Thus, PMEA can increase some natural immunity functions, such as NK activity and IFN production. These results suggest that PMEA might be active in vivo against HIV and herpes viruses both as an immunomodulator and as an antiviral compound.

Suppressor cells induced by influenza virus inhibit interleukin-2 production in mice.

PR8 virus depressed interleukin-2 (IL-2) and natural killer (NK) cell activity in BALB/c infected mice. IL-2 production was not dependent on (i) a decreased number of T cells or (ii) a primary defect in IL-1 production, but on a T-suppressor cell subpopulation. In fact, when T suppressor cells were removed from infected spleen cells, we observed normal levels of IL-2 activity.

Pinealectomy inhibits interleukin-2 production and natural killer activity in mice.

Four--five-week-old C57BL/6 mice were surgically pinealectomized. At different time intervals after surgery their spleens were removed and assayed for interleukin-2 (IL-2) production and natural killer (NK) cell activity. Non-operated and sham-operated mice were used as controls. The present results indicate that pinealectomy significantly reduced IL-2 production and NK cell activity, in comparison to sham-operated mice. These effects seem to be related to the lack of melatonin. In fact the subcutaneous injection of this hormone (50 or 100 mg/kg at 5 p.m.) in pinealectomized mice was able to restore IL-2 production and NK cell activity. However, chronic treatment with melatonin (10, 20 and 50 mg/kg for 9 consecutive days) failed to reverse the impairment of the immune responses.