Placental production of progestins is fully effective in villous cytotrophoblasts and increases with the syncytiotrophoblast formation.

Placental syncytiotrophoblast (ST) is considered as the main placental endocrine tissue secreting progesterone, a steroid essential for maintenance of pregnancy. However, each step of progestins production has been poorly investigated in villous cytotrophoblast (VCT) regarding ST formation. We aimed to characterize progestins production during human differentiation of VCT into ST. VCTs were isolated from term placenta and cultivated, with or without forskolin (FSK), to stimulate trophoblast differentiation. Secreted progestins concentrations were determined by immuno-assay and Gas Chromatography-tandem mass spectrometry. Intracellular expression of cholesterol transporter and enzymes involved in steroidogenesis were studied by immunofluorescence, western-blot, and RT-qPCR. Progesterone and pregnenolone are produced by VCT and their secretion increases with VCT differentiation while 17-hydroxyprogesterone concentration remains undetectable. HSD3B1 enzyme expression increases whereas MLN64, the cholesterol placental mitochondrial transporter and P450SCC expressions do not. FSK induces progestins production. Progestins placental synthesis is effective since VCT and increases with ST formation thanks to mitochondria.

[Interval between two smear tests for cervical cancer screening and characteristics of women with a short interval: Study in the department of Isère]. / Délai entre deux frottis de dépistage du cancer du col utérin et caractéristiques des femmes présentant un délai court : étude dans le département de l'Isère.

AIMS: In France, guidelines for cervical cancer screening recommend that women between the ages of 25-65 have a smear test performed once every three years. However, some women are screened significantly more frequently. In this study, we used a data-driven approach as opposed to a traditional hypothesis-driven approach to characterise the population of women who are screened more frequently than advised. METHODS: Data came from an organised cervical cancer screening programme of a French department in the Alps (Isère). We retrospectively selected women aged between 25 to 65 years old who had at least two smear tests during the follow up period (2011-2015). We used a data-driven clustering approach to compare the population of over-screened women with other populations. We then performed a descriptive analysis of the over-screened population using univariate (Chi2 test) and multivariate (logistic regression) methods. RESULTS: A total of 10,000 patients were randomly chosen from a population of 54,073. In our univariate analysis, women in the over-screened population were significantly younger, participated less in organised screening, were more likely to be followed by a gynaecologist and had more smear test results showing inflammation than the other populations. Patient location (urban v.s. rural area) was not significant for this population. The multivariate analysis confirmed these results. CONCLUSION: This data-driven approach based on an unsupervised learning method enables us to more accurately characterise the over-screened population. These data invite to improve communication with the youngest women and the gynecologists to recall the benefit of an interval between two normal smears complying with the recommendations. This approach could help to improve the prevention and have a real impact on this Public Health issue.

Carboplatin instead of cisplatin in combination with dexamethasone, high-dose cytarabine with or without rituximab (DHAC+/-R) is an effective treatment with low toxicity in Hodgkin's and non-Hodgkin's lymphomas.

The DHAP regimen (high-dose cytarabine in combination with dexamethasone and cisplatin) with or without rituximab (DHAP+/-R) is one of the most common regimens in daily practice. It is considered the standard treatment for relapse or refractory Hodgkin's and non-Hodgkin's lymphoma (NHL). Cisplatin nephrotoxicity is a major concern, and other platinum compounds are being tried. We performed a monocentric retrospective analysis to evaluate the use of carboplatin, so-called DHAC+/-R regimen. The purpose was to assess the toxicity of the DHAC+/-R regimen in real-life. The Dexamethasone, Cytarabine, Carboplatin (DHAC) regimen consisted of carboplatin AUC = 5 mg/ml/min (targeted area under the curve with Calvert's formula) on day 1, cytarabine 2 g/m2 twice a day on day 2 and IV dexamethasone 40 mg from days 1 to 4. Rituximab was administrated at 375 mg/m2 on day 1 for CD20+ NHL. The interval between courses was 21 days. During the period considered, 199 patients received DHAC+/-R. For the entire cohort, median follow-up is 24 months (range, 2-82), median OS is not reached (NR), estimated 2-year OS is 75% (95% CI, 69-83) and median progression-free survival (PFS) is 46 months (95% CI, 22-NA). Of 144 patients scheduled for autologous stem cell transplantation (ASCT), 102 (71%, NA = 2) were in response after DHAC+/-R and all except 4 underwent ASCT. Grade ≥ 3 haematological toxicities were mainly thrombocytopenia (n = 101) and anaemia (n = 95). Grade ≥ 3 neutropenia occurred in 10 patients. No grade ≥ 3 renal and one grade 3 neurological toxicity were reported. DHAC+/-R is feasible in daily practice, provides good response rates and jeopardises neither stem cell collection nor ASCT.

Lymphocyte expansion after unrelated cord blood allogeneic stem cell transplantation in adults.

Limited information is available regarding the incidence and features of lymphocyte expansions after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Large granular lymphocytes (LGL) expansions have been reported after bone marrow or peripheral blood, but not after unrelated cord blood (UCB) allo-HSCT, associated with indolent clinical courses and favorable outcomes. Here, we considered 85 recipients of UCB allo-HSCT to more broadly define the impact of lymphocytosis, not limited to LGL. Sustained lymphocytosis was observed in 21 (25%) patients at a median onset of 12.6 months and with a median duration of 12 months. Immunophenotypic analysis showed predominantly CD8+ T and/or polyclonal B-cell expansions. Three patients only had monoclonal T-cell expansion. CMV reactivation was significantly more frequent in the group of patients with lymphocytosis (76% vs 28%, P=0.0001), but was not associated with survival. Conversely, 2-year disease-free survival and overall survival were significantly higher for lymphocytosis patients (85% vs 55%, P=0.01 and 85% vs 63%, P=0.03, respectively). In conclusion, expansion of T or B lymphocytes after UCB allo-HSCT in adults is not a rare event. Although occurring relatively late after transplant, this feature is predictive of a better outcome for the patients.

Development of a Double-Antigen Microsphere Immunoassay for Simultaneous Group and Serotype Detection of Bluetongue Virus Antibodies.

Bluetongue viruses (BTV) are arboviruses responsible for infections in ruminants. The confirmation of BTV infections is based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs) using the BTV viral protein 7 (VP7) as antigen. The determination of the BTV serotype by serological analyses could be only performed by neutralization tests (VNT) which are time-consuming and require BSL3 facilities. VP2 protein is considered the major serotype-defining protein of BTV. To improve the serological characterization of BTV infections, the recombinant VP7 and BTV serotype 8 (BTV-8) VP2 were synthesized using insect cells expression system. The purified antigens were covalently bound to fluorescent beads and then assayed with 822 characterized ruminant sera from BTV vaccinations or infections in a duplex microsphere immunoassay (MIA). The revelation step of this serological duplex assay was performed with biotinylated antigens instead of antispecies conjugates to use it on different ruminant species. The results demonstrated that MIA detected the anti-VP7 antibodies with a high specificity as well as a competitive ELISA approved for BTV diagnosis, with a better efficiency for the early detection of the anti-VP7 antibodies. The VP2 MIA results showed that this technology is also an alternative to VNT for BTV diagnosis. Comparisons between the VP2 MIA and VNT results showed that VNT detects the anti-VP2 antibodies in an early stage and that the VP2 MIA is as specific as VNT. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple BTV serotypes can be detected simultaneously.

Rituximab maintenance after autologous stem cell transplantation prolongs response duration in non-naive rituximab follicular lymphoma patients: a single institution experience.

We retrospectively evaluated the role of rituximab (R) in maintenance treatment after autologous stem cell transplantation performed in patients with relapsed follicular lymphoma. We compared the outcome of 67 follicular lymphoma (FL) patients according to the use of rituximab maintenance (RM) or not. All patients received rituximab plus chemotherapy before autologous stem-cell transplantation (ASCT). Patients received median of two lines of prior therapy. The RM schedule was one injection of rituximab every 3 months for 2 years. Median follow-up is 4.6 years. The 3-year progression-free survival (PFS) after ASCT was 86 % with RM vs. 46 % without (p = 0.0045). Median is not reached in the RM arm vs. 31 months in non-RM arm. The 3-year OS was 96 % with RM vs. 78 % without (p = 0.059). The present monocentric study shows that 2 years of RM after ASCT significantly increases response duration for non-naive rituximab relapsed FL patients compared with observation.

Autologous hematopoietic stem cell transplantation in elderly patients (≥ 70 years) with non-Hodgkin's lymphoma: A French Society of Bone Marrow Transplantation and Cellular Therapy retrospective study.

INTRODUCTION: Limited data is available on the feasibility of high-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (AHSCT) in elderly patients over 70 years of age with non-Hodgkin's lymphoma (NHL). MATERIALS AND METHODS: In the setting of the Société Française de Greffe de Moelle et de Thérapie Cellulaire (SFGM-TC) group, we retrospectively analyzed 81 consecutive patients with NHL over 70 years of age who received AHSCT. RESULTS: The median age at AHSCT was 72.3 years [70-80]. Patients' were diagnosed with diffuse large B-cell lymphoma (n=40), follicular lymphoma (n=16), mantle cell lymphoma (n=15), T-cell lymphoma (n=5), and other (n=5). Hematopoietic Cell Transplantation Comorbidity Index (HCT-CI) was 0 in 73% of patients. Main conditionings were BEAM (Carmustine-Etoposide-Cytarabine-Melphalan, n=61) and melphalan alone (n=14). Median delays to reach 0.5×109/L neutrophils and 20 × 10(9)/L platelets were of 12 [9-76] days and 12 [0-143] days, respectively. One hundred day and one year cumulative incidence of NRM was 5.4% and 8.5%, respectively. The main cause of death remains relapse. CONCLUSION: In conclusion, this study revealed that AHSCT seemed to be acceptable in patients over 70 years of age with NHL. Patient age is not a limiting factor if clinical condition is adequate.

[Laparoscopic transposition of lower polar vessels for pyelo-ureteral junction obstruction: preliminary experience]. / Transposition des vaisseaux polaires pour syndrome de la jonction pyélo-urétérale : expérience préliminaire.

AIM OF THE STUDY: Lower pole vessels are a classical cause of PUJO even in children. The "gold standard" in the management of PUJ obstructions remains a dismembered pyeloplasty as described by Anderson and Hynes. However, some authors have developed an alternative procedure to this approach with encouraging results. The aim of our study was to evaluate our preliminary results concerning laparoscopic vascular hitch for crossing vessels. MATERIAL AND METHODS: We conducted a retrospective, monocentric study of all patients managed by this technique from January 2010 to December 2012. RESULTS: Eleven patients (7 boys, 4 girls) were managed by laparoscopy at a mean age of 10.7 years (5.4-17). They were referred to our center for clinical symptoms (intermittent pain 7, high blood pressure 1, UTI 1), antenatal diagnosis or accidental discovery. Obstruction was confirmed by MAG3 nephrogram and the presence of obstructive vessels by tomodensitometry or MRI. Mean operative time was 90.2minutes (48-184). Seven patients over 11 were strictly managed by laparoscopic transposition of lower pole vessels. Four required a classical video-assisted dismembered pyeloplasty due to a potential intraluminal stenosis. The latter were suspected by a distension test with furosemid in all four cases. Mean follow-up was 12.9±3 months. Nine patients over 11 were totally non symptomatic, whereas 2 still present mild intermittent pain. In all cases, ultrasound scans show an improvement of the pelvic dilatation. CONCLUSION: Laparoscopic transposition of lower pole vessels is a suitable and feasible alternative for the management of obstructive PUJ. Our preliminary experience emphasizes the need for a precise preoperative selection of patients along with a per operative evaluation of the obstruction. Further experience seems required to improve our criteria in this indication. LEVEL OF EVIDENCE: 5.

Chimerism analysis in peripheral blood using indel quantitative real-time PCR is a useful tool to predict post-transplant relapse in acute leukemia.

Detection of increasing mixed chimerism (IMC) using standard PCR correlates with relapse after allo-SCT for acute leukemias (ALs). Quantitative real-time PCR of insertion/deletion polymorphism (indel qrtPCR) is a much more sensitive method, which can be performed on peripheral blood. We studied the significance of low increases of recipient cells (0.1%) detected by indel qrtPCR in a cohort of 89 transplants. We did not observe relapse among the 32 patients with no IMC. Fifty-seven patients presented a first IMC, which was followed by four different scenarios: a decreasing MC (26 cases, no relapse), a stable MC (1 case, 1 relapse), a second IMC (24 cases, 15 relapse) or no control of chimerism (6 cases, 5 relapses). In multivariate analysis, detection of two successive IMCs was strongly associated with relapse (hazard ratio (HR): 9.4, 95% confidence interval (CI): 3.8-23; P<0.0001). Among the 57 patients who presented at least one IMC, 27 underwent immunomodulation (tapering of immunosuppression or donor lymphocyte injection), leading to a 1-year relapse rate of 15.7% vs 57.6% in the 30 other patients (P=0.0007). Altogether, these results indicate that chimerism analysis using indel qrtPCR in peripheral blood is a useful tool for detection of relapse in patients transplanted for AL.

[How I manage respiratory syncytial virus, human herpesvirus 6 and adenovirus reactivation or infection after allogeneic stem cell transplantation: a report of the SFGM-TC]. / Conduite à tenir devant une réactivation ou une infection à virus respiratoire syncytial, herpèsvirus 6 et adénovirus après allogreffe de cellules souches hématopoïétiques.

In the attempt to harmonize clinical practices between different French transplantation centers, the French Society of Bone Marrow Transplantation and Cell Therapy (SFGM-TC) set up the third annual series of workshops which brought together practitioners from all member centers and took place in October 2012 in Lille. Here we report our results and recommendations regarding the management of virus respiratory syncytial virus (RSV), human herpes virus 6 (HHV6) or adenovirus allogeneic Stem Cell Transplantation.

The natural history of clinical operational tolerance after kidney transplantation through twenty-seven cases.

We report here on a European cohort of 27 kidney transplant recipients displaying operational tolerance, compared to two cohorts of matched kidney transplant recipients under immunosuppression and patients who stopped immunosuppressive drugs and presented with rejection. We report that a lower proportion of operationally tolerant patients received induction therapy (52% without induction therapy vs. 78.3%[p = 0.0455] and 96.7%[p = 0.0001], respectively), a difference likely due to the higher proportion (18.5%) of HLA matched recipients in the tolerant cohort. These patients were also significantly older at the time of transplantation (p = 0.0211) and immunosuppression withdrawal (p = 0.0002) than recipients who rejected their graft after weaning. Finally, these patients were at lower risk of infectious disease. Among the 27 patients defined as operationally tolerant at the time of inclusion, 19 still display stable graft function (mean 9 ± 4 years after transplantation) whereas 30% presented slow deterioration of graft function. Six of these patients tested positive for pre-graft anti-HLA antibodies. Biopsy histology studies revealed an active immunologically driven mechanism for half of them, associated with DSA in the absence of C4d. This study suggests that operational tolerance can persist as a robust phenomenon, although eventual graft loss does occur in some patients, particularly in the setting of donor-specific alloantibody.

[Atypical onset of Churg and Strauss syndrome in a child]. / Révélation atypique d'un syndrome de Churg et Strauss chez l'enfant.

We report the case of a 13-year-old boy who had been treated since the age of 6 for moderate asthma. Except asthma, his past medical history was uneventful. The patient was referred for the sudden onset of bilateral leg edemas with peripheral purpuric lesions. Blood tests showed increased blood eosinophilia (9000/mm(3)) with no fever. The antineutrophil cytoplasmic antibodies (ANCA) were negative. The skin biopsy showed extensive ischemic subcutaneous necrosis related to necrotizing vasculitis. The general secondary symptoms occurred with multiorgan involvement (pulmonary infiltrates, peripheral neuropathy, gastrointestinal tract symptoms, and arthralgia). Genital infiltration was also noted. The child's general health was preserved. Neither cardiac nor renal involvement were found. The patient showed favorable clinical progression after oral prednisone therapy.

Colonoscopy practices, and colorectal cancer and polyp screening, as assessed in the French district of Isère from May to July in 2004.

BACKGROUND: A pilot program of organized screening for colorectal cancers was conducted in Isère, an administrative district in France. A fecal occult blood test (Hemoccult II(®)) was proposed for all individuals aged greater than 50 years (women since 1991 and men since 2002), followed by colonoscopy for those testing positive. A prospective study was carried out from May to July in 2004 and compared with a similar study conducted in 1996. The goal was to investigate colonoscopy practices, especially the role of screening. METHODS: Gastroenterologists practising in Isère (n=39/42 practitioners) completed a questionnaire including their patients' age and gender, indications, methods and results for all colonoscopies performed in those aged greater than 20 years. Any tissue samples taken were sent away for histological evaluation. RESULTS: The study involved 2558 colonoscopies (54% female, 73% patients aged greater or equal to 50 years), an increase of 35% from 1996 to 2004. Of the patients referred, 50.0% were symptomatic (pain; bowel problems: 28.7%; rectal bleeding: 21.3%), 23.5% had colonic disease and 22.5% came from screening (3.1% had positive stool tests, 17.8% had a family history). Recommendations related to family history (update of the 1998 consensus conference: screening indicated for patients with a first-degree relative diagnosed with cancer or advanced polyps aged less than 60 years) were well applied in terms of relatedness (81%) but, in 52% of cases, the age was greater than 60 years. Colonoscopy was carried out in almost all cases (0.1% failure), with complications in 0.4% of the examinations. Of the 2558 colonoscopies performed, 10% revealed advanced polyps or cancer: 30% were following a positive test compared with 8% for symptoms and 6% with a family history. Multivariate analyses showed that polyps greater or equal to 10mm or malignant tumors are 1.5 times more common in men than in women, and six times more frequently seen in patients having colonoscopy following a positive test for blood in stools than in those with a family history of colorectal cancer. The number of pathologies found increased significantly in those aged greater than 50 years. CONCLUSION: This cross-sectional survey of colonoscopy practices in Isère shows an increase in the number of colonoscopies performed between 1996 and 2004. This increase is not explained by expansion of the screening program, which was the reason for only 3% of colonoscopies. However, the best diagnostic yield for advanced polyps or cancers was obtained in screened patients (30%).

[Evaluation of the follow-up of women aged 50-74 years after cervical cytological Ascus abnormalities in cancer screening: adherence to clinical practice guidelines in Isere, France; 1991-2000]. / Evaluation de la prise en charge des femmes de 50 à 74 ans après frottis du col de l'utérus répondu Ascus dans un dépistage organisé en Isère 1991-2000 : application de la conduite à tenir de l'Agence nationale d'accréditation et d'évaluation en santé.

OBJECTIVES: The aim of this study is to compare to the guideline (1998 and 2001) the follow-up of Ascus cytological abnormalities among women aged 50-74 years who have participated at the combined breast, cervical and colorectal cancer screening programme from 1991 to 2000 in Isère, France. PATIENTS AND METHODS: The follow-up of 1154 women with Ascus smear was analysed. A woman was defined according follow-up if she have made a colposcopy or biopsy less than four months after one positive smear or if she has repeated three smears: 3-7 months and 10-14 months after the positive smear and 1 year after the last negative smear. RESULTS: The follow-up was according to guidelines for 28.4% of the 1154 women (150 women are unknowns), 58.6% had a follow-up with too long delay and 17.2% had an uncompleted follow-up. The follow-up did not differ before 1998. It did not differ from women age. Women who were treated by gynaecologist (548) had a better follow-up (according: 35.4%) than the women who were treated by a general practitioner (595). DISCUSSION AND CONCLUSION: The follow-up of Ascus cytological abnormalities is not according to guideline. The follow-up in the screening program will be intensified.

[Sensitivity and specificity of the breast screening program in the Isere region based on positive results between 1991 and 1999]. / Analyse de la sensibilité et de la spécificité des mammographies en fonction des taux de tests positifs dans le programme de dépistage organisé de l'Isère de 1991 à 1999.

PURPOSE: The screening program guidelines specify that the call back rate of women for additional imaging (positive mammogram) should not exceed 7% at initial screening, and 5% at subsequent screening. Materials and methods. Results in the Isere region (12%) have prompted a review of the correlation between the call back rate and indicators of quality (detection rate, sensitivity, specificity, positive predictive value) for the radiologists providing interpretations during that time period. RESULTS: Three groups of radiologists were identified: the group with call back rate of 10% achieved the best results (sensitivity: 92%, detection rate: 0.53%, specificity: 90%). The group with lowest call back rate (7.7%) showed insufficient sensitivity (58%). The last group with call back rate of 18.3%, showed no improvement in sensitivity (82%) and detection rate (0.53%), but showed reduced specificity (82%). CONCLUSION: The protocol update in 2001 does not resolve this problematic situation and national results continue to demonstrate a high percentage of positive screening mammograms. A significant increase in the number of positive screening examinations compared to recommended guidelines is not advantageous and leads to an overall decrease in the quality of the screening.

Differential effects of thyroid hormones on energy metabolism of rat slow- and fast-twitch muscles.

Thyroid hormone (TH) is an important regulator of mitochondrial content and activity. As mitochondrial content and properties differ depending on muscle-type, we compared mitochondrial regulation and biogenesis by T3 in slow-twitch oxidative (soleus) and fast-twitch mixed muscle (plantaris). Male Wistar rats were treated for 21 to 27 days with T3 (200 microg/kg/day). Oxidative capacity, regulation of mitochondrial respiration by substrates and phosphate acceptors, and transcription factors were studied. In soleus, T3 treatment increased maximal oxygen consumption (Vmax) and the activities of citrate synthase (CS) and cytochrome oxidase (COX) by 100%, 45%, and 71%, respectively (P < 0.001), whereas in plantaris only Vmax increased, by 39% (P < 0.01). ADP-independent respiration rate was increased in soleus muscle by 216% suggesting mitochondrial uncoupling. Mitochondrial substrate utilization in soleus was also influenced by T3, as were mitochondrial enzymes. Lactate dehydrogenase (LDH) activity was elevated in soleus and plantaris by 63% and 11%, respectively (P < 0.01), and soleus creatine kinase was increased by 48% (P < 0.001). T3 increased the mRNA content of the transcriptional co-activator of mitochondrial genes, PGC-1alpha, and the I and IV COX subunits in soleus. The muscle specific response to thyroid hormones could be explained by a lower content of TH receptors in plantaris than soleus. Moreover, TRalpha mRNA level decreased further after T3 treatment. These results demonstrate that TH has a major effect on mitochondrial content, regulation and coupling in slow oxidative muscle, but to a lesser extent in fast muscle, due to the high expression of TH receptors and PGC-1alpha transcription factor.

Scale-up of a myoblast culture process.

The effects of different types of cell carriers, strategies for cell transfer on carriers, and of several fusion inhibitors on the growth kinetics of primary human myoblasts culture were studied in order to develop a bioprocess suitable for the treatment of Duchenne muscular dystrophy based on the transplantation of unfused cells. Our results indicate that myoblast production is larger on Cytodex 1 and 3 than on polypropylene or polyester fabrics and on a commercial porous macrocarrier. Myoblast growth conditions with Cytodex 1 were further investigated to establish the bioprocess operating conditions. It was found that microcarrier density of 3 g DW l(-1), inoculum density of 2x10(5) cells ml(-1), and continuous agitation speed of 30-rpm result in final myoblast production comparable to static cultures. However, for all the culture conditions used, myoblasts growth kinetics exhibited a lag phase that lasted a minimum of 1 week prior to growth, the end of the lag phase correlating with the appearance of microcarrier aggregates. Based on this observation, we propose that aggregation promotes cell growth by offering a network of very large inter-particular pores that protect cells from mechanical stress. We took advantage of the presence of these aggregates for the scale-up of the culture process. Indeed, using myoblast-loaded microcarrier-aggregates instead of myoblast suspension to inoculate a fresh suspension of microcarriers significantly reduced the duration of the lag phase and allowed the scale-up of the bioprocess at the 500-ml scale. In order to ensure the production of unfused myoblasts, the efficiency of five different fusion inhibitors was investigated. Only calpeptin (9.1 microg ml(-1)) significantly inhibited the fusion of the myoblasts, while TGFbeta (50 ng ml(-1)) and LPA (10 microg ml(-1)) increased myoblasts growth but did not affect fusion, sphingosine (30 microg ml(-1)) induced a 50% death and NMMA (25 microg ml(-1)) had no effect on either growth or fusion. Finally, transplantation trials on severe combined immunodeficient mice showed that microcarrier-cultured human myoblasts grown using the optimized bioprocess resulted in grafts as successful as myoblasts grown in static cultures. The bioprocess, therefore, prove to be suitable for the large-scale production of myoblasts required for muscular dystrophy treatment.