Three-dimensional changes of a porcine collagen matrix and free gingival grafts for soft tissue augmentation to increase the width of keratinized tissue around dental implants: a randomized controlled clinical study.

BACKGROUND: Emerging clinical data points to the relevance of the presence of keratinized tissue (KT). Although apically positioned flap/vestibuloplasty along with free gingival graft (FGG) is considered as a standard intervention for augmenting KT, substitute materials appear to be a viable treatment alternative. So far, there is a lack of data investigating the dimensional changes at implant sites treated with soft-tissue substitutes or FGG. AIM: The present study aimed at comparing three-dimensional changes of a porcine derived collagen matrix (CM) and FGG for increasing KT at dental implants over a 6-month follow-up period. MATERIALS AND METHODS: The study enrolled 32 patients exhibiting deficient KT width (i.e., < 2 mm) at the vestibular aspect who underwent soft tissue augmentation using either CM (15 patients/23 implants) or FGG (17 patients/31 implants). The primary outcome was defined as tissue thickness change (mm) at treated implant sites between 1- (S0), 3- (S1), and 6-months (S2). Secondary outcomes considered changes of KT width over a 6-month follow-up period, surgical treatment time, and patient-reported outcomes. RESULTS: Dimensional analyses from S0 to S1 and from S0 to S2 revealed a mean decrease in tissue thickness of - 0.14 ± 0.27 mm and - 0.04 ± 0.40 mm in the CM group, and - 0.08 ± 0.29 mm and - 0.13 ± 0.23 mm in the FGG group, with no significant differences noted between the groups (3 months: p = 0.542, 6 months: p = 0.659). Likewise, a comparable tissue thickness decrease was observed from S1 to S2 in both groups (CM: - 0.03 ± 0.22 mm, FGG: - 0.06 ± 0.14 mm; p = 0.467). The FGG group exhibited a significantly greater KT gain after 1, 3 and 6 months compared to the CM group (1 month: CM: 3.66 ± 1.67 mm, FGG: 5.90 ± 1.58 mm; p = 0.002; 3 months: CM: 2.22 ± 1.44; FGG: 4.91 ± 1.55; p = 0.0457; 6 months: CM: 1.45 ± 1.13 mm, FGG: 4.52 ± 1.40 mm; p < 0.1). Surgery time (CM: 23.33 ± 7.04 min.; FGG: 39.25 ± 10.64 min.; p = 0.001) and postoperative intake of analgesics were significantly lower in the CM group (CM: 1.2 ± 1.08 tablets; FGG: 5.64 ± 6.39 tablets; p = 0.001). CONCLUSIONS: CM and FGG were associated with comparable three-dimensional thickness changes between 1 and 6 months. While a wider KT band could be established with FGG, the use of CM significantly reduced surgical time and patients´ intake of analgesics.

Single-cell transcriptomic atlas-guided development of CAR-T cells for the treatment of acute myeloid leukemia.

Chimeric antigen receptor T cells (CAR-T cells) have emerged as a powerful treatment option for individuals with B cell malignancies but have yet to achieve success in treating acute myeloid leukemia (AML) due to a lack of safe targets. Here we leveraged an atlas of publicly available RNA-sequencing data of over 500,000 single cells from 15 individuals with AML and tissue from 9 healthy individuals for prediction of target antigens that are expressed on malignant cells but lacking on healthy cells, including T cells. Aided by this high-resolution, single-cell expression approach, we computationally identify colony-stimulating factor 1 receptor and cluster of differentiation 86 as targets for CAR-T cell therapy in AML. Functional validation of these established CAR-T cells shows robust in vitro and in vivo efficacy in cell line- and human-derived AML models with minimal off-target toxicity toward relevant healthy human tissues. This provides a strong rationale for further clinical development.

Informing the development of a decision aid: Expectations and wishes from service users and psychiatrists towards a decision aid for antipsychotics in the inpatient setting.

OBJECTIVES: Decision aids (DAs) are promising tools to foster evidence-based shared decision-making between practitioners and service users. Nevertheless, it is still obscure how an evidence-based DA for people with severe mental illness, especially psychosis, should look in an inpatient treatment setting to be useful and feasible. Therefore, we conducted focus groups with psychiatrists and service users to collect and assess their expectations and wishes regarding an evidence-based DA. From these findings, we derived immediate recommendations for the future development of DAs. METHODS: We held two group interviews with service users (n = 8) and three group interviews with psychiatrists (n = 10). We used an open, large-scale topic guide. First, we presented data from a current meta-analysis on antipsychotics to the interviewees and, in a second step, asked for their expectations and wishes towards a DA that integrates these data. RESULTS: Our thematic analysis revealed six key themes addressed by the respondents: (1) general considerations on the importance and usefulness of such a DA, (2) critical comments on psychiatry and psychopharmacotherapy, (3) communicative prerequisites for the use of a DA, (4) form and content of the DA, (5) data input, data processing and output as well as (6) application of the DA and possible obstacles. CONCLUSIONS: Participants identified several important features for the development of DAs for selecting antipsychotics in inpatient psychiatric treatment. The digital format was met with the greatest approval. Especially the adaptability to different needs, users and psychopathologies and the possibility to outsource information dissemination via app seemed to be a decisive convincing argument. Further research is required to test specific features of DAs to be developed in clinical settings.

In vivo dynamics and anti-tumor effects of EpCAM-directed CAR T-cells against brain metastases from lung cancer.

Lung cancer patients are at risk for brain metastases and often succumb to their intracranial disease. Chimeric Antigen Receptor (CAR) T-cells emerged as a powerful cell-based immunotherapy for hematological malignancies; however, it remains unclear whether CAR T-cells represent a viable therapy for brain metastases. Here, we established a syngeneic orthotopic cerebral metastasis model in mice by combining a chronic cranial window with repetitive intracerebral two-photon laser scanning-microscopy. This approach enabled in vivo-characterization of fluorescent CAR T-cells and tumor cells on a single-cell level over weeks. Intraparenchymal injection of Lewis lung carcinoma cells (expressing the tumor cell-antigen EpCAM) was performed, and EpCAM-directed CAR T-cells were injected either intravenously or into the adjacent brain parenchyma. In mice receiving EpCAM-directed CAR T-cells intravenously, we neither observed substantial CAR T-cell accumulation within the tumor nor relevant anti-tumor effects. Local CAR T-cell injection, however, resulted in intratumoral CAR T-cell accumulation compared to controls treated with T-cells lacking a CAR. This finding was accompanied by reduced tumorous growth as determined per in vivo-microscopy and immunofluorescence of excised brains and also translated into prolonged survival. However, the intratumoral number of EpCAM-directed CAR T-cells decreased during the observation period, pointing toward insufficient persistence. No CNS-specific or systemic toxicities of EpCAM-directed CAR T-cells were observed in our fully immunocompetent model. Collectively, our findings indicate that locally (but not intravenously) injected CAR T-cells may safely induce relevant anti-tumor effects in brain metastases from lung cancer. Strategies improving the intratumoral CAR T-cell persistence may further boost the therapeutic success.

Analysis of immune responses in patients with CLL after heterologous COVID-19 vaccination.

Patients with chronic lymphocytic leukemia (CLL) treated with B-cell pathway inhibitors and anti-CD20 antibodies exhibit low humoral response rates following SARS-CoV-2 vaccination. To investigate this observation, a prospective single-institution study was conducted comparing peripheral blood mononuclear cell transcriptional response with antibody and T-cell response rates following heterologous BNT162b2/ChAdOx1 vaccination of 15 patients with CLL/small lymphocytic lymphoma (SLL). Two-dose antibody response rate was 40%, increasing to 53% after booster. Patients on Bruton tyrosine kinase inhibitor (BTKi) and venetoclax ± anti-CD20 antibody within 12 months of vaccination responded inferiorly compared with those under BTKi alone. The 2-dose-T-cell response rate was 80%, which increased to 93% after the booster dose. Key transcriptional findings were that interferon-mediated signaling activation including activation of the JAK-STAT pathway generally occurred within days of vaccination, but was independent from the magnitude of the antibody response. Increasing counts of IGHV genes were associated with B-cell reconstitution and improved humoral response rate in the vaccinated patients. T-cell responses in patients with CLL appeared independent of treatment status, whereas higher humoral response rate was associated with BTKi treatment and B-cell reconstitution. Boosting was particularly effective when intrinsic immune status was improved by CLL treatment. Limitations included studying a relatively small cohort, with different treatments and vaccination schedules.

Esthetic and clinical outcomes after immediate placement and restoration: Comparison of two implant systems in the anterior maxilla-A cross-sectional study.

AIM: To assess the esthetic and clinical performance of a novel self-tapping implant system for single-tooth restorations in the esthetic zone after immediate placement and provisionalization. MATERIALS AND METHODS: This cross-sectional study included 52 patients contributing a total of 52 immediately placed and restored implants with ≥12 months after functional loading, comparing two different implant systems: Straumann® BLX (Institut Straumann AG, Basel, Switzerland; 25 patients) and Ankylos® (Dentsply Sirona, Hanau, Germany; 27 patients). As the primary outcome measure, peri-implant tissue esthetics were assessed by means of pink esthetics score (PES) rated by three independent clinicians. Moreover, as secondary outcome measures, the peri-implant tissue health was assessed by means of bleeding on probing, probing depth, and suppuration. Apart from that, the modified plaque index, keratinized mucosa width, and the presence of mucosal recessions were also assessed. When clinical signs suggested the possibility of peri-implantitis, radiographs were indicated to assess progressive bone loss. RESULTS: The mean PES ratings were 12.10 ± 1.10 for Ankylos versus 11.2 ± 1.86 for BLX, both achieving good esthetic results without significant differences (p = 0.143). There were no differences among most clinical parameters (plaque, bleeding on probing, probing depth, peri-implant mucosal recession), although peri-implant mucositis was present in one-third of the cases. The inter-rater agreement on esthetics was not significant (p < 0.250). CONCLUSION: Within the limitations of the present study, it was concluded that the use of either BLX or Ankylos implant systems was associated to comparable peri-implant health and good pink esthetic outcomes during immediate implantation and restoration protocols, for at least 12 months.

Immediate versus delayed implant placement in the esthetic zone: a prospective 3D volumetric assessment of peri-implant tissue stability.

PURPOSE: To evaluate the volumetric stability of peri-implant soft and hard tissue prospectively, this study compared immediate versus delayed implants placed in the anterior esthetic region. METHODS: This non-randomized controlled clinical study included 25 patients, who received an immediate (type 1) or a delayed (type 4) implant placement for the replacement of a single anterior tooth. The anterior maxillae were intraorally scanned at three timepoints: before surgery (S0), 6 months (S1), and 12 months (S2) after surgery. A specific region of interest (ROI), divided into marginal and apical regions, was determined and superimposed for volumetric changes analysis. At 6 and 12 months, the probing depth (PD), bleeding/suppuration on probing (BOP/SUP), modified plaque index (PI), keratinized mucosa (KM) width, mucosal recession (MR), and implant stability (PTV) by means of periotest were recorded. RESULTS: Between S0-S2, tissue surrounding immediate implants was reduced in 0.37 ± 0.31 mm, whereas delayed implants gained 0.84 ± 0.57 mm mean tissue volume. Peri-implant tissue loss at type 1 implants occurred primarily in the marginal section of the ROI (0.42 ± 0.31 mm), whereas tissue gain at type 4 implants occurred mainly in the apical section (0.83 ± 0.51 mm). These values were significantly different between both groups for the entire ROI (p = 0.0452) and the marginal region (p = 0.0274). In addition, the mean buccal KM width around type 1 implants was significantly wider in comparison with the type 4 implants group after 12 months (p = 0.046). There were no significant differences between groups regarding PD, BOP/SUP, or PTV. CONCLUSIONS: The results suggest that type 1 implants placed in the esthetic region experience more tissue loss than type 4 implants, thus marginal tissue remodeling should be considered for planning immediate implants placement in the anterior maxillae.

Accumulation of mutations in antibody and CD8 T cell epitopes in a B cell depleted lymphoma patient with chronic SARS-CoV-2 infection.

Antibodies against the spike protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can drive adaptive evolution in immunocompromised patients with chronic infection. Here we longitudinally analyze SARS-CoV-2 sequences in a B cell-depleted, lymphoma patient with chronic, ultimately fatal infection, and identify three mutations in the spike protein that dampen convalescent plasma-mediated neutralization of SARS-CoV-2. Additionally, four mutations emerge in non-spike regions encoding three CD8 T cell epitopes, including one nucleoprotein epitope affected by two mutations. Recognition of each mutant peptide by CD8 T cells from convalescent donors is reduced compared to its ancestral peptide, with additive effects resulting from double mutations. Querying public SARS-CoV-2 sequences shows that these mutations have independently emerged as homoplasies in circulating lineages. Our data thus suggest that potential impacts of CD8 T cells on SARS-CoV-2 mutations, at least in those with humoral immunodeficiency, warrant further investigation to inform on vaccine design.

Comprehensive analysis of immune responses in CLL patients after heterologous COVID-19 vaccination.

Patients with chronic lymphocytic leukemia (CLL) treated with B-cell pathway inhibitors and anti-CD20 antibodies exhibit low humoral response rate (RR) following SARS-CoV-2 vaccination. To investigate the relationship between the initial transcriptional response to vaccination with ensuing B and T cell immune responses, we performed a comprehensive immune transcriptome analysis flanked by antibody and T cell assays in peripheral blood prospectively collected from 15 CLL/SLL patients vaccinated with heterologous BNT162b2/ChAdOx1 with follow up at a single institution. The two-dose antibody RR was 40% increasing to 53% after booster. Patients on BTKi, venetoclax ± anti-CD20 antibody within 12 months of vaccination responded less well than those under BTKi alone. The two-dose T cell RR was 80% increasing to 93% after booster. Transcriptome studies revealed that seven patients showed interferon-mediated signaling activation within 2 days and one at 7 days after vaccination. Increasing counts of COVID-19 specific IGHV genes correlated with B-cell reconstitution and improved humoral RR. T cell responses in CLL patients appeared after vaccination regardless of treatment status. A higher humoral RR was associated with BTKi treatment and B-cell reconstitution. Boosting was particularly effective when intrinsic immune status was improved by CLL-treatment.

Volumetric tissue changes following combined surgical therapy of peri-implantitis: A 2-year follow-up analysis. A prospective case series.

AIM: To assess volumetric tissue changes following combined surgical therapy of peri-implantitis over a follow-up period of 24 months. MATERIALS AND METHODS: A total of 20 patients (n = 28 implants) were diagnosed with peri-implantitis and underwent access flap surgery, implantoplasty, and augmentative therapy at intrabony components (ie, combined therapy) using a natural bone mineral and a native collagen membrane. The peri-implant region of interest (ROI) was intraorally scanned pre-operatively (S0), and after 12 (S3) and 24 (S4) months. Digital files were superimposed for the assessment of volumetric changes between the referred time points. The change in thickness was assessed at a standardized ROI, segmented into two equidistant sections (ie, marginal and apical). RESULTS: Peri-implant tissues exhibited a nonsignificant mean thickness loss of 0.16 (95% CI: -4 to 0.06) and 0.17 mm (95% CI: -0.05 to 0.4) at S3 and S4, respectively. S0-S3 dimensional thickness changes at marginal and apical areas were -0.24 (95% CI: -0.48 to 0.002) and -0.19 mm (95% CI: -0.36 to -0.2), respectively. Dimensional changes from S0 to S4 amounted to -0.22 mm (95% CI: -0.46 to 0.02) and -0.07 mm (95% CI: -0.09 to 0.2), respectively. The thickness changes at marginal and apical ROIs were significant from S0 to S3. Clinical parameters (ie, plaque index, bleeding on probing, and probing depth) significantly improved over the 24-month follow-up period. Linear regression analyses revealed no significant association between baseline bone loss (%), width of keratinized mucosa, and mucosal recession scores and thickness changes. CONCLUSIONS: Peri-implant tissues revealed minor volumetric changes at 12 and 24 months after combined surgical therapy.

The role of preoperative albumin-bilirubin grade for oncological risk stratification in liver transplant patients with hepatocellular carcinoma.

BACKGROUND AND OBJECTIVES: Albumin-bilirubin (ALBI) score was shown to correlate with liver function and tumor recurrence after hepatectomy for hepatocellular carcinoma (HCC). The aim of this study was to assess the prognostic value of ALBI grade in liver transplantation (LT) patients with HCC. METHODS: Pre-LT available independent predictors of recurrence-free survival (RFS) and microvascular tumor invasion (MVI) were determined in 123 patients with HCC. RESULTS: Posttransplant HCC recurrence rates were 10.5%, 15.9%, and 68.2% in ALBI grade 1, 2, and 3, respectively (P < .001). Along with serum α-fetoprotein (AFP) and C-reactive protein (CRP) levels, ALBI grades 1 or 2 was identified as an independent predictor of RFS (hazard ratio, 3.52; 95% confidence interval [CI], 1.577-7.842; P = .002). Furthermore, ALBI grade 3 proved to be the strongest indicator of MVI (odds ratio, 11.59; 95% CI, 3.412-39.381; P < .001). A novel oncological risk score-based on AFP, CRP, and ALBI grade provided the best discriminative capacity (c-statistic 0.806) in selecting liver recipients with low oncological risk profile. CONCLUSION: Preoperative ALBI grade seems to be valuable for refinement of oncological risk stratification at LT for HCC.

Firefly Luciferase-Based Reporter Gene Assay for Investigating Nanoparticle-Mediated Nucleic Acid Delivery.

Investigation of nanoparticle-mediated nucleic acid delivery is a key step for the development of nucleic acid-based nanotherapeutics. Using luciferases as reporter genes, the efficiency of gene delivery can be quantified in a highly sensitive way based on bioluminescence measurements. Here we describe a robust assay to quantify the activity of exogenously produced firefly luciferase and its normalization by the total protein amount (bicinchoninic acid assay, BCA) in the cells. The method describes preparation of firefly luciferase assay buffer (F-LAB) along with BCA assay and employment of the optimized firefly luciferase assay for investigating in vitro gene delivery by polyplex and lipoplex nanoparticles. Reusability of F-LAB for ease of usage (by freezing and reusing it for luciferase assay) is also demonstrated.

Prognostic relevance of RIP140 and ERß expression in unifocal versus multifocal breast cancers: a preliminary report.

The aim of this study was to investigate the expression of two nuclear receptor transcriptional coregulators, namely RIP140 (receptor-interacting protein of 140 kDa) and LCoR (ligand-dependent corepressor) in unifocal versus multifocal breast cancers. The expression of these two proteins was analyzed by immunohistochemistry in a matched-pair cohort of 21 unifocal and 21 multifocal breast tumors. The expression of the two estrogen receptors (ERα and ERß) was studied in parallel. RIP140 and LCoR levels appeared lower in unifocal tumors compared to multifocal samples (decreased of immune-reactive scores and reduced number of high expressing cells). In both tumor types, RIP140 and LCoR expression was correlated with each other and with expression of ERß. Very interestingly, the expression of RIP140, LCoR, and ERß was inversely correlated with overall survival only for the unifocal group. The negative correlation with overall and recurrence free survival was more pronounced in patients whose unifocal tumors expressed high levels of both RIP140 and ERß. Altogether, this preliminary report indicates that the ERß/RIP140 signaling is altered in unifocal breast cancers and correlated with patient outcome. Further investigation is needed to decipher the molecular mechanisms and the biological relevance of this deregulation.

[Psycho-oncology in dermatological practice : Evaluation of need and care structures]. / Psychoonkologie in dermatologischen Praxen : Bedarfseinschätzung und Versorgungsstrukturen.

BACKGROUND: Psycho-oncological care is a main component of comprehensive oncological care as stated in the National Cancer Plan of the German Federal Government. Correspondingly this goal has been adopted in the strategy of the German Skin Cancer Council. In certified skin cancer centers structural requirements for psycho-oncological care are established. Nevertheless, a large proportion of skin cancer patients are treated in dermatological practices. Up to now data on psycho-oncological care in dermatological practices are missing. MATERIALS UND METHODS: We conducted a descriptive cross-sectional written survey on psycho-oncological care in dermatological practices from October 2016 to February 2017. RESULTS: In all, 171 practices completed the questionnaire; 19.4% of these practices have an oncological focus. The mean number of treated skin cancer patients was 554.3 ± 659.1 and 62.4 ± 73.6 for melanoma patients. Dermatologists estimated a low proportion (≤5%) of patients with need for psycho-oncological care; however, 21.9% of practices actively offer information on psycho-oncological programs and 26.1% cooperate with psycho-oncological care providers. Interest in psycho-oncological care concepts was stated by 29.3%. CONCLUSIONS: Psycho-oncological care is only occasionally and partly deficiently provided in dermatological practices including referral to psycho-oncological care professionals. The results emphasize the necessity to raise awareness regarding psycho-oncological needs of skin cancer patients and to integrate psycho-oncological counselling into clinical routine in dermatological practices.

Importance of RIP140 and LCoR Sub-Cellular Localization for Their Association With Breast Cancer Aggressiveness and Patient Survival.

New markers are needed to improve diagnosis and to personalize treatments for patients with breast cancer (BC). Receptor-interacting protein of 140 kDa (RIP140) and ligand-dependent corepressor (LCoR), two transcriptional co-regulators of estrogen receptors, strongly interact in BC cells. Although their role in cancer progression has been outlined in the last few years, their function in BC has not been elucidated yet. In this study, we investigated RIP140 and LCoR localization (cytoplasm vs nucleus) in BC samples from a well-characterized cohort of patients (n = 320). RIP140 and LCoR were expressed in more than 80% of tumors, (predominantly in the cytoplasm), and the two markers were highly correlated. Expression of RIP140 and LCoR in the nucleus was negatively correlated with tumor size. Conversely, RIP140 and LCoR cytoplasmic expression strongly correlated with expression of two tumor aggressiveness markers: N-cadherin and CD133 (epithelial mesenchymal transition and cancer stem cell markers, respectively). Finally, high RIP140 nuclear expression was significantly correlated with longer overall survival, whereas high total or cytoplasmic expression of RIP140 was associated with shorter disease-free survival. Our study strongly suggests that the role of RIP140 and LCoR in BC progression could vary according to their prevalent sub-cellular localization, with opposite prognostic values for nuclear and cytoplasmic expression. The involvement in BC progression/invasiveness of cytoplasmic RIP140 could be balanced by the anti-tumor action of nuclear RIP140, thus explaining the previous contradictory findings about its role in BC.

Precise redox-sensitive cleavage sites for improved bioactivity of siRNA lipopolyplexes.

Lipo-oligomers have been proven as potent siRNA carriers based on stable electrostatic and hydrophobic complex formation and endosomal membrane destabilization. Although high stability of siRNA polyplexes is desirable in the extracellular space and cellular uptake, intracellular disassembly is important for the cytosolic release of siRNA and RNA-induced silencing complex formation. To improve the release, bioreducible sequence-defined lipo-oligomers were synthesized by solid-phase assisted synthesis using the disulfide building block Fmoc-succinoyl-cystamine for precise positioning of a disulfide unit between a lipophilic diacyl (bis-myristyl, bis-stearyl or bis-cholestanyl) domain and an ionizable oligocationic siRNA binding unit. Reducible siRNA polyplexes show higher gene silencing efficacy and lower cytotoxicity than their stable analogs, consistent with glutathione-triggered siRNA release and reduced lytic activity.

EGF receptor targeted lipo-oligocation polyplexes for antitumoral siRNA and miRNA delivery.

Antitumoral siRNA and miRNA delivery was demonstrated by epidermal growth factor receptor (EGFR) targeted oligoaminoamide polyplexes. For this purpose, the T-shaped lipo-oligomer 454 was used to complex RNA into a core polyplex, which was subsequently functionalized with the targeting peptide ligand GE11 via a polyethylene glycol (PEG) linker. To this end, free cysteines on the surface of 454 polyplex were coupled with a maleimide-PEG-GE11 reagent (Mal-GE11). Resulting particles with sizes of 120-150 nm showed receptor-mediated uptake into EGFR-positive T24 bladder cancer cells, MDA-MB 231 breast cancer cells and Huh7 liver cancer cells. Furthermore, these formulations led to ligand-dependent gene silencing. RNA interference (RNAi) triggered antitumoral effects were observed for two different therapeutic RNAs, a miRNA-200c mimic or EG5 siRNA. Using polyplexes modified with a ratio of 0.8 molar equivalents of Mal-GE11, treatment of T24 or MDA-MB 231 cancer cells with miR-200c led to the expected decreased proliferation and migration, changes in cell cycle and enhanced sensitivity towards doxorubicin. Delivery of EG5 siRNA into Huh7 cells resulted in antitumoral activity with G2/M arrest, triggered by loss of mitotic spindle separation and formation of mono-astral spindles. These findings demonstrate the potential of GE11 ligand-containing RNAi polyplexes for cancer treatment.