Methods for inactivation of seafood Anisakis larvae and prevention of human anisakiasis: a mini-review.

Anisakiasis is an arising zoonosis induced by parasitic nematodes belonging to the family Anisakidae. Anisakiasis is often caused by the ingestion of larval nematodes in uncooked or minimally processed seafood dishes, which are regularly consumed by humans. Significant potential sources of infection are raw fish (e.g., sushi and sashimi) that can be found in traditional Japanese cuisine and can be part of the culinary tradition of consumption of raw or marinated fish that is particularly diffused in European countries. During the last five decades, the global prevalence of human anisakiasis has been rising, becoming an emergent major public health problem. Thus, there is an unmet need for well-defined and cost-effective methods aimed at killing Anisakis larvae, thus reducing the incidence of anisakiasis. In this mini-review, we discuss the clinical features of anisakiasis as well as the effectiveness and mechanisms of action of the main methods employed for increasing seafood safety and killing Anisakis larvae, including freezing, heating, use of high hydrostatic pressure, salting process, pepsin digestion method and use of garlic oil.

In vitro activity of fosfomycin against mucoid and non-mucoid Pseudomonas aeruginosa strains.

OBJECTIVES: Pseudomonas aeruginosa is the most frequent infectious agent in cystic fibrosis patients. P. aeruginosa resistance to first line antibiotics limits therapeutic options, but the therapeutic potential of older generation antibiotics, such as fosfomycin is under investigation. Fosfomycin does not belong to any other antibiotic class and acts by inhibiting the biosynthesis of the bacterial cell wall during the initial phases. A major problem for the use of fosfomycin against P. aeruginosa is the absence of a clinical breakpoint, the last one of 32 µg/mL was proposed in 2013 by the CA-SFM (Comité de l'Antibiogramme de la Société Française de Microbiologie). METHODS: Sixty-one strains of P. aeruginosa (thirty mucoid and thirty-one non mucoid) were collected from respiratory samples of cystic fibrosis patients. All isolates were identified by MALDI-TOF (Bruker, Bremen, Germany). Fosfomycin MICs against P. aeruginosa were measured using an automated system and confirmed by the gold standard method. RESULTS: There was no significant difference between mucoid and non-mucoid strains. MIC distribution and susceptibility rates were obtained by agar dilution method and from this data we measured MIC50 and MIC90 which were equal to 32 µg/mL and 64 µg/mL, respectively. From automated method results we measured a very major error (VME), major error (ME) and categorical agreement (CA) which were equal to 0%, 11% and 89%, respectively. Comparing automated and agar dilution methods, a Cohen's kappa equal to 73% (0.726) was measured. CONCLUSIONS: Our data suggest that fosfomycin has good effect against mucoid and non-mucoid strains of P. aeruginosa and automated systems can be implemented in clinical microbiology laboratories to assess fosfomycin with rapid and reproducible results.

COS-7-based model: methodological approach to study John Cunningham virus replication cycle.

John Cunningham virus (JCV) is a human neurotropic polyomavirus whose replication in the Central Nervous System (SNC) induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV propagation and PML investigation have been severely hampered by the lack of an animal model and cell culture systems to propagate JCV have been very limited in their availability and robustness. We previously confirmed that JCV CY strain efficiently replicated in COS-7 cells as demonstrated by the progressive increase of viral load by quantitative PCR (Q-PCR) during the time of transfection and that archetypal regulatory structure was maintained, although two characteristic point mutations were detected during the viral cycle. This short report is an important extension of our previous efforts in defining our reliable model culture system able to support a productive JCV infection.Supernatants collected from transfected cells have been used to infect freshly seeded COS-7 cell line. An infectious viral progeny was obtained as confirmed by Western blot and immunofluorescence assay. During infection, the archetype regulatory region was conserved.Importantly, in this study we developed an improved culture system to obtain a large scale production of JC virus in order to study the genetic features, the biology and the pathogenic mechanisms of JC virus that induce PML.

A multi-scale approach to colorectal cancer: from a biochemical- interaction signaling-network level, to multi-cellular dynamics of malignant transformation. Interplay with mutations and onco-protein inhibitor drugs.

This review article is part of a special Current Cancer Drug Targets issue devoted to colorectal cancer and molecularly targeted treatments. In our paper we made an attempt to connect more basic aspects with preclinical, pharmacological / therapeutic and clinical aspects. Reconstruction of a Molecular Interaction Map (MIM) comprising an important part of the G0 - G1 - S cell cycle transition, was a major component of our review. Such a MIM serves also as a convenient / organized database of a large set of important molecular events. The frequency of mutated / altered signaling-proteins indicates the importance of this signaling-network region. We have considered problems at different scale levels. Our MIM works at a biochemical-interaction level. We have also touched the multi-cellular dynamics of normal and aberrant colon crypts. Until recently, dynamic simulations at a biochemical or multi-cellular scale level were considered as a sort of esoteric approach. We tried to convince the reader, also on the basis of a rapidly growing literature, mostly published in high quality journals, that suspicion towards simulations should dissipate, as the limitations and advantages of their application are better appreciated, opening the door to their permanent adoption in everyday research. What is really required is a more interdisciplinary mentality and an interdisciplinary approach. The prize is a level of understanding going beyond mere intuition.

Antioxidant biomarkers and food intake in elderly women.

OBJECTIVE: To investigate the relationship between antioxidant biomarkers and food intake in elderly women. DESIGN: Cross-sectional study. SETTING: Recreation Center for the Elderly in the city of Itajaí, Santa Catarina, Brazil. PARTICIPANTS: 73 elderly women with an average age of 71 years, 93% caucasian, average body weight 68.7 ± 13.1 kg and average BMI 28.5 ± 2.3 kg/m². MEASUREMENTS: Nutritional status was assessed based on the Body Mass Index (BMI). Data on food intake were obtained by applying the 24h diet recall method in three non-consecutive days, including Sunday. The assessment of antioxidant biomarkers was performed based on tests for total plasma thiols and phenolic compounds. The linear regression analysis was used to assess the effect of the consumption of food groups on antioxidant biomarkers. RESULTS: A positive association was found between thiols and intake of carotenoid-rich vegetables (p=0.03), oils, fats and oilseeds (p=0.03); a negative association was observed between total concentrations of phenolic compounds and intake of cereals (p=0.04). CONCLUSION: The intake of foods from the carotenoid-rich vegetables, oils, fats and oilseeds food groups increased the levels of plasma thiols, and the intake of foods from the group of cereals decreased the plasma concentration of phenols. Studies should be conducted to investigate the association between the intake of antioxidant-rich foods and the plasma antioxidant profile, as a way to protect against the aging process.

Factors associated with oxidative stress in women with breast cancer.

OBJECTIVE: To assess the association between physiological, physical, lifestyle and nutritional variables and oxidative stress biomarkers in women with breast cancer. METHODS: This cross-sectional study was conducted on 55 women newly diagnosed with breast cancer. The extent of oxidative stress was analyzed by the measurement of plasma lipid hydroperoxides (LH), thiobarbituric acid reactive substances (TBARS), protein carbonyl, whole blood reduced glutathione (GSH) and serum antioxidant capacity (AC). Diet data were obtained from food frequency questionnaire. Linear regression was used to determine the association between the variables studied and oxidative stress biomarkers. The protein carbonyl data was not included in the linear regression analyses since the data did not show a normal distribution, even after logarithmic and other transformations. RESULTS: After adjusting for energy intake, the intake of chicken and high-fat dairy products was associated with increased levels of LH, while vitamin E intake was associated with decreased LH levels (R² = 23.8%). Intake of oils was associated with increased levels of TBARS (R² = 6.82%). Positive axillary lymph node status was associated with decreased levels of GSH (R² = 9.31%). Increasing age was directly associated with levels of AC, while animal fat, dairy product, and sweet food intakes were associated with low levels of AC (R² = 41.42%). CONCLUSION: Intake of chicken, vitamin E, dairy products (particularly high-fat dairy products), oils, animal fat, and sweet foods, along with axillary lymph node status and age, may be important determinants of oxidative stress in women with breast cancer.

Dietary intake and oxidative stress in breast cancer: before and after treatments.

OBJECTIVE: The aim of this study was to investigate changes in dietary intake, anthropometric parameters and markers of oxidative stress in 40 women who underwent surgery, chemotherapy or radiation therapy for breast cancer. METHODS: Pretreatment and post-treatment measurements included data collected through a food frequency questionnaire, weight and height to calculate the body mass index (BMI) and oxidative stress markers assessed from blood reduced glutathione (GSH), serum antioxidant capacity (AC), plasma thiobarbituric acid reactive substances (TBARS), serum lipid hydroperoxides (LH) and plasma carbonyls. Differences were compared using paired Student's t-test or paired Wilcoxon's test. RESULTS: A significant increase (P < 0.05) in the intake of the food groups: meat and eggs, dairy products, beans, oils and fats, as well as food from the subgroups: red meat, milk and other dairy products rich in fat, fruit rich in vitamin C and vegetable fats was found after treatments. There was a significant increase in body weight (P < 0.05), BMI (P < 0.05), levels of TBARS (P < 0.0001), LH (P < 0.005) and carbonyls (P < 0.0001) and a significant decrease of levels of AC (P < 0.005) and GSH (P < 0.0001). CONCLUSION: Breast cancer diagnosis and treatments were associated with dietary intake changes and increased body weight, BMI and oxidative stress. These potential changes have important implications for preventive nutrition counseling.

Perinatal outcome and neurological follow up of the cotwins in twin pregnancies complicated by single intrauterine death.

OBJECTIVE: To review the outcome of twin pregnancies complicated by single fetal intrauterine death (IUD) managed at our Centre and to evaluate the neurological follow up of the surviving cotwins. STUDY DESIGN: Twenty-three twin pregnancies (10 dichorionic and 13 monochorionic diamniotic) complicated by IUD in the II or III trimester were seen at our Centre during the study period (2001-2006). All patients were managed conservatively unless non-reassuring signs of fetal well-being were present at ultrasound examination or CTG after 28 weeks, suggesting immediate delivery. Serial scans after the diagnosis of single death were performed and, in addition, eight monochorionic twin pregnancies underwent prenatal MRI in order to identify the presence of cerebral lesions in the survivors. Live born surviving cotwins underwent neurological follow up. RESULTS: In the monochorionic group one cotwin died in utero and one in the neonatal period with a perinatal survival rate of 83.4% (10/12) (excluding one case who opted for termination of pregnancy); in the dichorionic group perinatal survival rate was 100%. In all monochorionic cases there were no signs of ischemic brain lesions in the surviving cotwins at the diagnosis of single death and during ultrasonographic follow up. In monochorionic pregnancies prenatal MRI, when performed, was negative for signs of brain damage in the surviving cotwins. Gestational age at delivery was not statistically different between monochorionic and dichorionic pregnancies (36 (range, 28.4-40.2) vs. 34.6 (range, 28.2-41.3) weeks) (p=0.6) and the rate of early preterm delivery before 32 weeks was 23.8% (5/21) and independent from chorionicity (18.2% vs. 30%, p=0.5). Neurodevelopmental follow up was available for 18/20 live born survivors (85%) and was normal in all but one twin; this case was born from a dichorionic pregnancy with a suspicion of congenital infection. CONCLUSIONS: Our data confirmed a trend to a higher risk of perinatal mortality of cotwins in monochorionic twin pregnancies compared to dichorionic ones. In our experience prenatal ultrasound and MRI were useful to exclude cerebral lesions in utero and subsequent neurological sequelae in surviving monochorionic cotwins, even if definitive conclusions, especially on MRI, are limited by the small number of cases in our study.

Severe brain damage from twin-twin transfusion syndrome treated with serial amnioreductions after 26 weeks: a case to reconsider the gestational age limits of laser therapy.

We report a case of monochorionic twin pregnancy complicated by twin-twin transfusion syndrome (TTTS) diagnosed in the late second trimester and treated with two amnioreductions. Three days after the first amniodrainage, the recipient twin developed intracranial ventriculomegaly and, similarly, after a few days, the donor showed signs of brain damage at MRI. We discuss the possible mechanism of brain damage of amnioreductions performed after 26 weeks of gestation in a monochorionic pregnancy with TTTS as a result of a placental 'steal' phenomenon.

Post-operative remnants of brainstem cavernomas: incidence, risk factors and management.

INTRODUCTION: The risk of leaving a remnant after surgery for a cavernous malformation in the brainstem is generally not stressed enough, even though such remnants appear to have a high risk of re-bleeding. At least 40% of known cavernoma remnants after surgery have further bleeding episodes. A retrospective analysis of 30 patients with brainstem cavernoma who underwent surgery is presented, focusing on incidence, risk factors and management of post-surgical residuals. The sites were, medulla in three patients, pons-medulla in four, pons in 16, pons-midbrain in four and midbrain in three. All 30 patients came to our clinical observation with at least one episode of acute-onset neurological deficit and all were operated in the sub-acute phase. Only one patient had a worse stable outcome than the pre-surgical state, and 29 did better or were stable. All patients had a brain MRI scan within 72 h after surgery to confirm that complete removal had been achieved. In three, although the surgical cavity and its border appeared clean at the end of surgery, with no lesion remaining, post-operative MRI detected a residuum. These three patients were re-operated, but one had a further bleed prior to excision. MATERIALS AND METHODS: In our series, the surgical finding of a multi-lobular cavernoma (as opposed to the more frequent finding of a discrete lesion with a thick capsule), with a thin wall and satellite nodules separated by a thin layer of apparently intact white matter, was common (seven patients). This group included the three patients with evidence of residuum on post-operative MRI. In our experience, the surgical finding of a multi-lobular cavernoma carries a higher risk of residuum and post-surgical re-bleeding. CONCLUSION: Immediate post-operative brain MRI scans are therefore strongly recommended for their detection, especially in this group of patients, and if a residual is detected early re-intervention is less risky than the natural history.

HMGA molecules in neuroblastic tumors.

The high mobility group A (HMGA) proteins are thought to work as ancillary transcription factors and to regulate the expression of a growing number of genes through direct binding to DNA or via protein-protein interactions. Both HMGA1 and HMGA2 are important regulators of basic biological processes, including cell growth, differentiation and transformation. Their qualitatively or quantitatively altered expression has been described in a number of human tumors. We studied and review here their expression in neuroblastic tumors. HMGA2 is expressed only in a subset of ex vivo neuroblastoma (NB) tumors and in the embryonic adrenal gland, but it is undetectable in the adult adrenal gland, suggesting that its anomalous expression might be associated with NB tumorigenesis and/or tumor progression. In vitro, its expression is easily detectable in retinoic acid (RA)-resistant cell lines. The exogenous expression of HMGA2 is sufficient to convert RA-sensitive SY5Y NB cells into RA-resistant cells, thus suggesting that HMGA2 might be a relevant player in determining NB cell responses to endogenous or therapeutically important growth inhibitory substances. In contrast, HMGA1 expression is readily detectable in all NB cell lines and tumors, but its expression is consistently higher in less differentiated NBs compared with ganglioneuromas and ganglioneuroblastomas. Interestingly, RA increases HMGA1 expression in RA-resistant NB cells but inhibits it in cells undergoing RA-induced growth inhibition and neuronal differentiation. Our studies indicate that HMGA molecules might be biologically and pathologically relevant factors in neuroblastic tumor development and progression.

[Role of cerebral magnetic resonance angiography in childhood]. / Ruolo dell'angiografia a risonanza magnetica cerebrale in età pediatrica.

This study evaluates the role of cerebral MR Angiography (MRA), as a part of the flowchart for the assessment of the brain in childhood. This noninvasive technique provides intra- and extra-cranial anatomic and physiologic vascular information, with a level of precision almost equal to that of arterial angiography. Indeed, MRA has reached a level of diagnostic accuracy such that the need for arterial angiography can be avoided, in most cases of cerebrovascular disorder. Therefore arterial angiography could be performed at the subacute (and more neurologically stable) stage, or presurgically, yielding additional morphologic and hemodynamic insights into cerebrovascular disease. MRA is also indicated at follow-up examination, and in treatment planning.

Pseudobactin biogenesis in the plant growth-promoting rhizobacterium Pseudomonas strain B10: identification and functional analysis of the L-ornithine N(5)-oxygenase (psbA) gene.

Pseudobactin(B10), the fluorescent siderophore produced by the rhizobacterium Pseudomonas strain B10, contains the hydroxamate ligand D-N(5)-hydroxyornithine (D-N(5)-OH-Orn). We cloned the L-Orn N(5)-oxygenase (psbA) gene from a genomic library of Pseudomonas strain B10 and demonstrated that PsbA is involved in the conversion of L-Orn to its N(5)-OH derivative. PsbA shows significant similarity to microbial omega-amino acid hydroxylases containing flavin adenine dinucleotide and NADP cofactor-binding sites and the FATGY signature of the putative substrate recognition pocket. The psbA gene is monocistronic, and its transcription is negatively controlled by iron. A site-specific psbA mutant of Pseudomonas strain B10 was biochemically complemented with the precursor L-N(5)-OH-Orn, suggesting that L-Orn is hydroxylated before conversion to the D isomer. The L-Orn N(5)-hydroxylase-defective mutants of Pseudomonas strain B10 and Pseudomonas aeruginosa PAO1 were much less effective than the parental strains in suppressing the growth of the phytopathogen Erwinia carotovora in iron-poor medium. The extent of in vitro inhibition of E. carotovora was strictly iron dependent and directly correlated with the amount of released siderophores. These data strengthen the role of fluorescent siderophores in biocontrol of deleterious rhizomicroorganisms.

Percutaneous radio-frequency thermal ablation of nonresectable hepatocellular carcinoma after occlusion of tumor blood supply.

PURPOSE: To evaluate the usefulness of percutaneous radio-frequency (RF) thermal ablation of nonresectable hepatocellular carcinoma (HCC) after occlusion of the tumor arterial supply. MATERIALS AND METHODS: Sixty-two patients with cirrhosis and biopsy-proved HCC underwent RF ablation after interruption of the tumor arterial supply by means of occlusion of either the hepatic artery with a balloon catheter (40 patients) or the feeding arteries with gelatin sponge particles (22 patients). RESULTS: After a single RF procedure in 56 patients and after two procedures in six patients, spiral computed tomography (CT) demonstrated a nonenhancing area corresponding in shape to the previously identified HCC, which was suggestive of complete necrosis. No major complications occurred. Two patients subsequently underwent surgical resection; the remaining 60 patients were followed up with spiral CT. During a mean follow-up of 12.1 months, 11 HCC nodules showed areas of local progression; 49 were identified as nonenhancing areas with a 40%-75% reduction in maximum diameter. The 1-year estimate of failure risk was 19% for local recurrence and 45% for overall intrahepatic recurrence. The estimated 1-year survival was 87%. Histopathologic analysis of one autopsy and two surgical specimens revealed more than 90% necrosis in one specimen and 100% necrosis in two. CONCLUSION: HCC nodules 3.5-8.5 cm in diameter can be ablated in one or two RF sessions after occlusion of the tumor arterial supply.

The pathology of human West Nile Virus infection.

West Nile Virus (WNV) was identified by immunohistochemistry (IHC) and polymerase chain reaction (PCR) as the etiologic agent in 4 encephalitis fatalities in New York City in the late summer of 1999. The fatalities occurred in persons with a mean age of 81.5 years, each of whom had underlying medical problems. Cardinal clinical manifestations included fever and profound muscle weakness. Autopsy disclosed encephalitis in 2 instances and meningoencephalitis in the remaining 2. The inflammation was mostly mononuclear and formed microglial nodules and perivascular clusters in the white and gray matter. The brainstem, particularly the medulla, was involved most extensively. In 2 brains, cranial nerve roots had endoneural mononuclear inflammation. In addition, 1 person had acute pancreatitis. Based on our experience, we offer recommendations for the autopsy evaluation of suspected WNV fatalities.

A case of Buerger's disease.

Buerger's disease (thromboangiitis obliterans) is an uncommon vascular occlusive disease most commonly affecting the lower extremities of young male tobacco smokers. We report the case of a thirty-five-year-old man who presented with nonhealing ulcerations of several toes. Arteriography revealed severe bilateral vascular occlusion. The patient failed to respond to intensive topical care, nifedipine, pentoxifylline, hyperbaric oxygen, intravenous antibiotics, and whirlpool. He continued to smoke, and eventually became septic, requiring amputations of his gangrenous left second toe and right leg below the knee.

[Factors associated with breast feeding. II Study of women with no prenatal control]. / Factores asociados a la practica de lactancia II. Estudio de mujeres sin control prenatal.

PIP: This prospective study on breastfeeding practices sought to identify if factors that intervene or determine breastfeeding practices are those influenced by women themselves, promoted by institutions such as hospitals or is a combination of both factors. The methodology included 582 women who did not attend prenatal consultations and only attended the 3 hospitals included in the study to deliver their babies. Hospital A studied 252 women, supported breastfeeding practices and gave patients milk samples; hospital B studied 80 women, did not support breastfeeding and gave milk samples; and hospital C interviewed 250 women, did not support breastfeeding and did not give women milk samples. Invitations to attend the study explained that 12 hours postpartum a questionnaire would be given and 15 days postpartum a session would be held to verify if they were breastfeeding. The independent variables were: place of birth, residence of the new mothers and that their own mothers age, educational level, status of relationship with the father of the baby, number of pregnancies, number of live children, background and number of previous stillbirths, were they living with the father of the baby and was he supporting her, was the pregnancy planned, previous problems with breastfeeding, was previous baby breastfed number of previous babies breastfed, reasons for not breastfeeding, and prior to the study what were other sources of information on breastfeeding. Results demonstrated that the sociocultural and demographic differences between each of the 3 populations attending the 3 hospitals were found to have stronger effects on breastfeeding practices than hospital policies.^ieng

Factores asociados a la práctica de la lactancia: II. Estudio de mujeres sin control prenatal / Factors associated with breast feeding: II. study of women with no prenatal control

Se revisan los casos sin control prenatal de un estudio prospectivo sobre práticas de la lactancia. La finalidad es identificar con la mayor precisión factores que intervienen o determinan las prácticas de alimentación infantil dentro de la que interesa destacar las diferencias que se encuentran entre instituciones con diferentes esquemas de atención. Con el fin último de evaluar si las características distintivas que se encuentran son atribuibles a las mujeres y su entorno o a las instituciones y sus políticas o, lo que es más probable, algún tipo de interacción entre ambos donde, se espera una mayor determinación social y una menor institucional. Se presentas la información de 582 mujeres que sólo acudieron a la atención del parto en tres instituciones de salud: promoción de la lactancia y no dotación de sucedáneos de leche (Hospital A: 252 mujeres); no promoción de la lactancia y dotación de sucedáneos de leche (Hospital B: 80 mujeres); y no promoción de la lactancia y no dotación de sucedáneos de leche (Hospital C: 250 mujeres). En el análisis, se encontró que el hospital A presentó: la menor proporción de madres que viven actualmente en un azona rural; la mayor proporción de mujeres con niveles más altos de escolaridad (secundaria o más); sus compañeros mostraron la menor frecuencia de analfabetos o con niveles más bajos de educación (analfabetos, primaria completa); este mismo comportamiento lo presentaron sus madres; la mayor proporción de mujeres con más de una muerte intrauterina y también mostró el mayor número de mujeres que no lactaron a su bebé anterior; por otro lado en el hospital B se encontró la mayor proporción de mujeres que sí viven con el papá del bebé y las que más reciben ayuda econímica por parte de él y el mayor número de mujeres que no recibieron nuevas ideas con la información recibida fuera de la institución; para finalizar, el hospital C msotró: la mayor proporción tanto de mujeres nacidas en un área rural como de madres analfabetas, fueron las mujeres que más recibieron información sobre la lactancia fuera de la institución. Es muy probable que las diferencias socioculturales y demográficas encontradas sean más delimitantes de la práctica que la influencia de la institución hospitalaria

[Muscular fixation of thallium-201 in lower limb arteriopathies before and after injection of Royat thermal gas]. / Etude de la fixation musculaire du thallium-201 dans les artériopathies des membres inférieurs avant et après injection de gaz thermal de Royat.

Thirty patients with peripheral vascular disease (grade II) were studied by way of thallium-201 (Tl-201) muscular scan in order to assess a possible muscular effect of the Royat spa natural gas. Immediately after a 1.1 MBq/kg Tl-201 IV injection, a dynamic scintigraphic study was performed during 15 minutes in order to obtain a time-activity curve (characterized by Tmax) over the thighs, at rest. Then static anterior and posterior views were performed over thighs and calves during 4 minutes each. Perfusion ratios were calculated as thigh/knee and calf/ankle. The GE maxi 400 T gamma-camera was connected to an Informatek treatment system, zones of interest being chosen to obtain anterior and posterior thigh/knee, leg/ankle uptake ratios. Two studies were realized: the first one in basal conditions, the second one 7 days later, immediately after a s/c injection of natural gas. The later study was preceded by a static counting to subtract the residual muscular activity. After gas injection, one observed that Tmax was shorter, on the average, and that the difference between time-activity curves observed during the first study disappeared for patients with unilateral disease. A significant Tl-201 uptake increase was observed for the anterior image of the calf, while no difference was noted on posterior images, natural gas being injected on the front face of legs. This was particularly clear for patients with unilateral disease and receiving only unilateral gas injection.