The cause of redetachment after vitrectomy with air tamponade for a cohort of 1715 patients with retinal detachment: an analysis of retinal breaks reopening.

BACKGROUND: To investigate the prevalence and predictors of retinal breaks reopening after vitrectomy with air tamponade in rhegmatogenous retinal detachment (RRD). METHODS: A retrospective cohort study was conducted in Shanghai General Hospital. Chart review was performed among 1715 patients with primary RRD who received pars plana vitrectomy (PPV) with air tamponade as initial management. Patients were followed up for recurrence. The clinical features of the eyes with retinal breaks reopening were recorded. Logistic regression was constructed to investigate the predictors for breaks reopening. RESULTS: A total of 137 (7.99%) patients had recurrent retinal detachment after PPV with air tamponade. The causes of surgery failure included new or missed retinal breaks (48.9%), reopening of original tears (43.8%) and proliferative vitreoretinopathy (7.3%). The median time to recurrence for the patients with breaks reopening was 18.0 days. Multivariate logistic regression indicated that the presence of retinal break(s) ≥ 1.5 disc diameters (DD) (odds ratio [OR]: 2.68, 95% confidence interval [CI]: 11.04-6.92, P = 0.041), and shorter period for restricted activities (OR: 0.94, 95% CI: 0.89-0.99, P = 0.020) were the independent predictors for breaks reopening. CONCLUSIONS: Breaks reopening is an important cause for retinal redetachment after PPV with air tamponade in primary RRD. The first 2-4 weeks after surgery is the "risk period" for breaks reopening. Special attention should be paid for patients with retinal break(s) ≥ 1.5 DD. A prolonged period for restricted activities is recommended.

Air versus Perfluoropropane Gas in Pars Plana Vitrectomy for Primary Rhegmatogenous Retinal Detachment: A 3-Year Retrospective Study.

INTRODUCTION: This study aimed to compare anatomical outcomes of air and perfluoropropane gas (C3F8) tamponade in pars plana vitrectomy for the treatment of rhegmatogenous retinal detachment (RRD). METHODS: In this retrospective study, data were gathered from 578 patients (578 eyes) with RRD. The follow-up records of all 578 patients that underwent primary vitrectomy for RRD with air or C3F8 were examined and analyzed. Surgical outcomes of the two groups were compared. RESULTS: A total of 342 eyes were treated with air and 236 with C3F8. The mean follow-up period was 37.65 ± 2.33 months. Baseline and preoperative clinical characteristics were similar between groups, but the period to intraocular bubble disappearance (p < 0.0001), intraocular pressure on the first postoperative day (p < 0.0001), number of cases with intraocular pressure >21 mm Hg within 3 days post-surgery (p < 0.0001), and the number with intraocular pressure >21 mm Hg during follow-up (p = 0.0002) differed significantly between groups. Primary reattachment rates for air and C3F8 groups were 95.03% and 95.34%, respectively. Clinical characteristics were similar in those with and without successful reattachment, and the frequency of new or unclosed breaks was similar between the two groups. There was no significant difference in two groups according to the presence or absence of inferior retinal breaks and inferior detached quadrants. Univariate and multivariate logistic regression identified no risk factor for surgical failure. CONCLUSIONS: Air showed equivalent effects to C3F8, with a shorter period to intraocular bubble disappearance, less risk of postoperative intraocular hypertension, and less expense.

Long-term surgical outcomes and prognostic factors of foveal detachment in pathologic myopia: based on the ATN classification.

BACKGROUND: To investigate the long-term surgical outcomes and prognostic factors of foveal detachment (FD) in pathological myopia. METHODS: This retrospective observational study included 59 patients with FD (61 eyes) who underwent pars plana vitrectomy at Shanghai General Hospital between June 2017 and July 2018 with follow-up for at least 24 months. Comprehensive ophthalmic examinations, including best-corrected visual acuity (BCVA) and swept-source optical coherence tomography, were assessed. Preoperative myopic maculopathy was evaluated according to the ATN classification. RESULTS: FD completely resolved in 59 of 61 eyes (96.7%). Mean duration of retinal reattachment was 12.10 ± 8.10 months. Mean logMAR BCVA improved from 1.34 ± 0.52 to 0.83 ± 0.43 at 24 months postoperatively (P < 0.001). Secondary macular hole occurred in 8 eyes (13.1%) with a mean period of 3.4 ± 4.1 weeks after primary surgery. In regression analyses, baseline myopic atrophy maculopathy (MAM) (B = 0.213, P = 0.005) and vitreomacular traction (VMT) (B = 0.292, P = 0.007) were adverse prognostic factors for postoperative BCVA. A more severe MAM revealed a delay in retinal reattachment (B = 5.670, P = 0.002). FD eyes with VMT (OR = 1.309, P = 0.003) or outer lamellar macular hole (O-LMH) (OR = 1.369, P < 0.001) were risk factors for postoperative secondary macular hole. CONCLUSIONS: Vitrectomy was effective in the long-term for treating FD. Careful consideration is needed for those with VMT or O-LMH due to the high risk of secondary macular hole after vitrectomy. FD eyes with more severe MAM tended to have poorer postoperative BCVA and extended periods of retinal reattachment.

A case of anti-VEGF therapy application in Takayasu arteries with retinopathy.

PURPOSE: Takayasu arteritis (TA) is a systemic granulomatous large vessel vasculitis that involves mainly the aorta and its primary branches, and occurs most commonly in young females. Ocular manifestations of TA include small vessels dilation, microaneurysm, arteriovenous anastomosis, retinal ischemia and retinopathy. However, no specific and effective treatments for Takayasu retinopathy is applied. This case aimed to demonstrate the role of anti-VEGF (vascular endothelial growth factor) therapy in treating Takayasu retinopathy. OBSERVATIONS: We herein reported an 18-year-old Asian woman who presented with typical wreath-like arteriovenous anastomosis around the disc in the right eye and vitreous hemorrhage in the left eye. The stenosis and occlusion of bilateral subclavian arteries, carotid arteries and other proximal arteries on angiography confirmed the diagnosis of TA. Meanwhile, elevated ESR and CRP revealed that TA was in the active stage. We applied anti-VEGF therapy in treating Takayasu retinopathy specially to inhibit neovascularization. Additionally, vitreous extraction was conducted in the left eye after the treatment of anti-VEGF therapy. CONCLUSIONS AND IMPORTANCE: This is the first report of effective application of anti-VEGF therapy in inhibiting wreath-like arteriovenous anastomosis and improving vitrectomy in TA.

Functional evaluation with microperimetry in large idiopathic macular holes treated by a free internal limiting membrane flap tamponade technique.

BACKGROUND: Free internal limiting membrane (ILM) flap tamponade technique is an alternative choice for treating large idiopathic macular holes (IMHs). However, the functional recovery related to this surgical approach is not well-characterized. This study aimed to evaluate morphological and microperimetric outcomes 6 months after free ILM flap tamponade technique for large IMHs. METHODS: Twenty-two patients (22 eyes) with large IMHs (minimal diameter > 400 µm) were retrospectively enrolled in this study. All patients underwent 23-gauge pars plana vitrectomy with ILM peeling and free ILM flap tamponade procedures. Snellen best-corrected visual acuity (BCVA), optical coherence tomography (OCT), and MP-1 microperimetry were measured at baseline and 6 months after surgery. Associations of postoperative BCVA with retinal sensitivity were detected. RESULTS: Macular hole closure was achieved in 21 eyes (95.5%). Dislodgement of free ILM flap was found in non-closed eye. Mean logMAR BCVA improved from 1.10 ± 0.33 at baseline to 0.67 ± 0.32 at 6 months postoperatively (P < 0.001). The mean overall macular sensitivity and foveal fixation stability increased respectively from 8.58 ± 3.05 dB and 65.64 ± 17.28% before surgery to 11.55 ± 2.72 dB and 78.59 ± 13.00% at 6 months after surgery (P < 0.001). The mean change in foveal sensitivity (within 2°) was significantly greater than the change achieved for peri-foveal sensitivity (2° to 10°) by 1.50 ± 2.62 dB (P = 0.014). Linear regression analysis showed that postoperative logMAR BCVA was significantly associated with duration of symptom (B = 0.063, P = 0.001), preoperative logMAR BCVA (B = 0.770, P = 0.000), preoperative peri-foveal (B = - 0.065, P = 0.000) and foveal sensitivity (B = - 0.129, P = 0.000). Moreover, multiple regression model revealed that preoperative foveal sensitivity was independently associated with postoperative logMAR BCVA (B = - 0.430, P = 0.040). CONCLUSIONS: Vitrectomy combined with ILM peeling and free ILM flap tamponade technique results in effective morphological and functional recovery for large IMHs. Preoperative foveal sensitivity might be a prognostic indicator for postoperative BCVA.

A cross-sectional study of vitreous and serum high mobility group box-1 levels in proliferative diabetic retinopathy.

PURPOSE: We determined vitreous and serum levels of high mobility group box-1 (HMGB-1) in patients with proliferative diabetic retinopathy (PDR) and elucidate their relationship with receptor for advanced glycation end products (RAGE), vascular endothelial growth factor (VEGF) and interleukin-1ß (IL-1ß). METHODS: In this cross-sectional study, patients with PDR who underwent vitrectomy were enrolled, and the control group included non-diabetic eyes. Vitreous and serum samples were analysed for HMGB-1, RAGE, VEGF and IL-1ß by ELISA. We investigated the correlation between serum and vitreous levels of each cytokine, and we analysed the influence of intravitreal anti-VEGF treatment prior to vitrectomy on the cytokine levels in PDR. RESULTS: Of 78 eyes of 78 patients enrolled consecutively, there were 32 PDR eyes and 46 control eyes. The serum levels were higher in diabetic than in non-diabetic subjects for HMGB-1, RAGE, VEGF and IL-1ß (all p < 0.001), respectively. Similarly, the vitreous levels were higher in diabetic than in non-diabetic subjects for HMGB-1 (p < 0.001), RAGE (p = 0.001), VEGF (p < 0.001) and IL-1ß (p < 0.001), respectively. We found a positive correlation between serum and vitreous levels of HMGB-1 in patient with PDR (p = 0.047, R = 0.353). There was a negative correlation between serum and vitreous levels of VEGF in patient with PDR (p = 0.001, R = -0.546). For the subgroup analysis, we detected that the vitreous levels of RAGE were significantly lower in patients who underwent anti-VEGF injection prior to vitrectomy than those who did not (p < 0.001). CONCLUSIONS: Our findings suggest that HMGB-1 is involved in PDR disorders, and it may be a novel therapeutic target to inhibit progression of PDR.

Metabolomic profile of diabetic retinopathy: a GC-TOFMS-based approach using vitreous and aqueous humor.

AIM: To identify the potential metabolite markers in diabetic retinopathy (DR) by using gas chromatography coupled with time-of-flight mass spectrometry (GC-TOFMS). METHODS: GC-TOFMS spectra were acquired from vitreous and aqueous humor (AH) samples of patients with DR and non-diabetic participants. Comparative analysis was used to elucidate the distinct metabolites of DR. Metabolic pathway was employed to explicate the metabolic reprogramming pathways involved in DR. Logistic regression and receiver-operating characteristic analyses were carried out to select and validate the biomarker metabolites and establish a therapeutic model. RESULTS: Comparative analysis showed a clear separation between disease and control groups. Eight differentiating metabolites from AH and 15 differentiating metabolites from vitreous were highlighted. Out of these 23 metabolites, 11 novel metabolites have not been detected previously. Pathway analysis identified nine pathways (three in AH and six in vitreous) as the major disturbed pathways associated with DR. The abnormal of gluconeogenesis, ascorbate-aldarate metabolism, valine-leucine-isoleucine biosynthesis, and arginine-proline metabolism might weigh the most in the development of DR. The AUC of the logistic regression model established by D-2,3-Dihydroxypropanoic acid, isocitric acid, fructose 6-phosphate, and L-Lactic acid in AH was 0.965. The AUC established by pyroglutamic acid and pyruvic acid in vitreous was 0.951. CONCLUSIONS: These findings have expanded our understanding of identified metabolites and revealed for the first time some novel metabolites in DR. These results may provide useful information to explore the mechanism and may eventually allow the development of metabolic biomarkers for prognosis and novel therapeutic strategies for the management of DR.

LncRNA TDRG1-Mediated Overexpression of VEGF Aggravated Retinal Microvascular Endothelial Cell Dysfunction in Diabetic Retinopathy.

PURPOSE: Diabetic retinopathy (DR), a neurovascular disease, is one of the leading causes of blindness in working-age adults. Long noncoding RNAs (lncRNAs) have attracted attention as indicators for DR. This study aimed to characterize the role of lncRNA human testis development-related gene 1 (TDRG1) and its modulation of vascular endothelial growth factor (VEGF) in deteriorating DR. METHODS: Tissue samples were obtained from patients with epiretinal membranes (EMs) or proliferative DR, and human retinal microvascular endothelial cells (HRECs) were cultured with high-glucose medium to mimic DR as the in vitro model. The expression of lncRNA TDRG1 and VEGF was determined by immunofluorescence staining, Western blotting, and RT-qPCR. Transfection of small-interfering RNA was conducted to knock down target gene expression. HREC functions were evaluated by cell viability, fluorescein isothiocyanate (FITC)-dextran extravasation, migration, and tube formation assays under different conditions. RESULTS: LncRNA TDRG1 and VEGF were found to be co-expressed and significantly upregulated in fibrovascular membranes (FVMs) from DR patients compared to those from EM patients. In the in vitro model, hyperglycemic treatment markedly increased the expression of lncRNA TDRG1 and VEGF at the mRNA and protein levels, which promoted cell proliferation and migration, enhanced permeability, and disrupted tube formation of HRECs. However, knockdown of lncRNA TDRG1 or VEGF notably decreased the expression of VEGF and reversed the impaired functions of high-glucose-treated HRECs. CONCLUSIONS: LncRNA TDRG1 promoted microvascular cell dysfunction via upregulating VEGF in the progression of DR and may serve as a potential therapeutic target in DR treatment.

Mechanism of MicroRNA-146a/Notch2 Signaling Regulating IL-6 in Graves Ophthalmopathy.

BACKGROUND/AIMS: We intended to investigate the significance of microRNA-146a, Notch2 and IL-6 on Graves ophthalmopathy (GO) and the relationships among them. METHODS: About 27 GO patients were incorporated in this study, including 13 patients with inactive GO and14 patients with active GO. Another 15 patients who had previously received strabismus orthopedics or ophthalmectomy due to trauma were selected as the control population. QRT-PCR assay was used to detect microRNA-146a and Notch2 expression levels in plasma. MTT assay and flow cytometry were respectively used to assess the viability and mitosis of the fibroblasts isolated from orbital connective tissue. Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was employed to detect serum IL-6 levels. The dual luciferase reporter gene assay was used to verify the targeting relationship between microRNA-146a and Notch2. RESULTS: Compared with the control group, the relative expression of miR-146a was significantly increased whereas the relative expression of Notch2 was significantly decreased (all P < 0.05) in GO patients compare with the control. Notch2 can be directly targeted by microRNA-146a. The over-expression of miR-146a markedly facilitated Orbital Fibroblasts (OFs) viability and mitosis whereas markedly suppressed cell apoptosis (all P < 0.05). Exogenous microRNA-146a mimics could down-regulat the expression of Notch2 and up-regulate IL-6 (P < 0.05). The inhibition of microRNA-146 resulted in the elevated expression of Notch2 and decreased expression of IL-6 (P < 0.05). CONCLUSION: MicroRNA-146a may increase the IL-6 levels and exacerbate GO by directly targeting Notch2.

Prevalence and predictors of metamorphopsia after successful rhegmatogenous retinal detachment surgery: a cross-sectional, comparative study.

AIMS: To evaluate the prevalence of metamorphopsia after successful rhegmatogenous retinal detachment (RRD) surgery and determine the independent predictors of metamorphopsia. METHODS: A cross-sectional, comparative study. The study included 380 eyes of 357 patients who underwent a successful RRD surgery between 2009 and 2015. The presence of metamorphopsia was tested with an Amsler grid and the foveal microstructure was scanned with high-resolution spectral-domain optical coherence tomography (OCT) at a follow-up visit. Medical records were reviewed for preoperative and intraoperative factors associated with postoperative visual recovery. RESULTS: The mean follow-up period was 35.1±19.6 months. The prevalence of metamorphopsia in our study was 46.58% (177/380), whereas in macula-off cases this number increased to 56.69% (144/254). Stepwise logistic regression analysis indicated that younger age at the diagnosis (OR=0.97, p<0.01), preoperative macula-off status (OR=3.34, p<0.01), postoperative presence of subretinal fluid (SRF) (OR=3.58, p=0.01) and disrupted external limiting membrane (ELM) junction (OR=1.79, p=0.02) were the independent predictors for metamorphopsia after surgery. The abnormal foveal microstructures after successful surgeries mainly included six types: disrupted inner segment/outer segment (133 eyes, 35.00%), disrupted ELM (131 eyes, 34.47%), epiretinal membrane (44 eyes, 11.58%), SRF (26 eyes, 6.84%), macular hole (9 eyes, 2.37%) and cystoid macular oedema (6 eyes, 1.58%). CONCLUSIONS: Considering metamorphopsia, special attention should be paid to young patients and/or the cases with involvement of macula. High-resolution OCT is informative in detecting anatomic abnormalities after RRD surgery. The postoperative presence of SRF and disrupted ELM line are independent predictors of metamorphopsia.

Optical Coherence Tomography Angiography Characteristics of Fovea in Residual Subfoveal Perfluorocarbon Liquid Eye.

The authors report a case of optical coherence tomography angiography (OCTA) features of retina and choriocapillaris before and after the surgical removal of a subfoveal perfluorocarbon liquid (PFCL) bubble. Before the surgery, the fovea avascular zone (FAZ) of both superficial and deep inner retinal layers (SRL/DRL) dramatically enlarged. However, after the removal, the FAZ continually decreased in both layers, even becoming close to normal in size. The abnormal choriocapillaris dark areas were also undetectable. Before and after surgery, the angle of the maximum FAZ diameter constantly remained horizontal and within normal limit in the SRL, forming an oval-shaped FAZ. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:1062-1066.].

Long-Term Follow-Up of the Fellow Eye in Patients Undergoing Surgery on One Eye for Treating Myopic Traction Maculopathy.

Objective. To observe the fellow eye in patients undergoing surgery on one eye for treating myopic traction maculopathy. Methods. 99 fellow eyes of consecutive patients who underwent unilateral surgery to treat MTM were retrospectively evaluated. All patients underwent thorough ophthalmologic examinations, including age, gender, duration of follow-up, refraction, axial length, intraocular pressure, lens status, presence/absence of a staphyloma, and best-corrected visual acuity (BCVA). Fundus photographs and SD-OCT images were obtained. When feasible, MP-1 microperimetry was performed to evaluate macular sensitivity and fixation stability. Results. At an average follow-up time of 24.7 months, 7% fellow eyes exhibited partial or complete MTM resolution, 68% stabilized, and 25% exhibited progression of MTM. Of the 38 eyes with "normal" macular structure on initial examination, 11% exhibited disease progression. The difference in progression rates in Groups 2, 3, and 4 was statistically significant. Refraction, axial length, the frequency of a posterior staphyloma, chorioretinal atrophy, initial BCVA, final BCVA, and retinal sensitivity all differed significantly among Groups 1-4. Conclusions. Long axial length, chorioretinal atrophy, a posterior staphyloma, and anterior traction contribute to MTM development. Patients with high myopia and unilateral MTM require regular OCT monitoring of the fellow eye to assess progression to myopic pre-MTM. For cases exhibiting one or more potential risk factors, early surgical intervention may maximize the visual outcomes.

Acute intraocular inflammation caused by endotoxin after intravitreal injection of counterfeit bevacizumab in Shanghai, China.

PURPOSE: To describe an outbreak of intraocular inflammation caused by endotoxin-contaminated counterfeit bevacizumab in China. DESIGN: Retrospective, observational case series. PARTICIPANTS: Patients undergoing intravitreal injection at a public hospital in September 2010. METHODS: The medical records and microbiology results of patients who presented with intraocular inflammation after injection with intravitreal counterfeit bevacizumab were reviewed. MAIN OUTCOME MEASURES: The incidence of intraocular inflammation, results of pathogen cultures, and clinical features of inflammation. RESULTS: A total of 116 patients (70 men and 46 women) were injected from 3 vials of counterfeit bevacizumab. Intraocular inflammation developed in 80 patients. The estimated median incubation period was 12 hours (range, 2-24 hours), and the median duration of symptoms was 6 days (range, 3-22 days). All patients were treated initially with topical corticosteroid and antibiotics. Vitreous tap and intravitreal injection were performed on 43 patients. Twenty-one patients with hypopyon and significant vitreous inflammation underwent vitrectomy. Microscopic evaluations and microbiologic cultures of all ocular specimens were negative for bacterial and fungal contamination. The presence of endotoxin in specimens was confirmed by laboratory testing. We refer to this new clinical syndrome as "endotoxin-induced ocular toxic syndrome" (EOTS). The inflammation regressed rapidly after treatment, and 63 patients (78.8%) recovered their pre-injection vision. CONCLUSIONS: This study implicates endotoxin as the cause of intraocular inflammation after the intravitreal injection of counterfeit bevacizumab. The EOTS appeared clinically distinct from typical infectious endophthalmitis.

[Proteomics analysis of serum biomarkers in patients with pathological myopia].

OBJECTIVE: To identify the correlation between serum-based differentially expressed proteins and pathological myopia. METHODS: It was a case-control study. The serum of 30 pathological myopia patients and 30 age- and gender-matched normal controls were collected from Shanghai First People's Hospital affiliated to Shanghai Jiaotong University. These patients were divided into 4 groups including macular-off retinal detachment (8 cases), retinal geographic atrophy (5 cases), macular hole (11 cases) and choroidal neovascular (6 cases). The serum specimens of normal controls were used as immunogen to immune rabbits in order to prepare polyclonal antibodies. Purified by Protein A cartridge, these mixed antibodies were then combined with CNBr-activated Sepharose so as to synthesize affinity medium which was finally used to treat the serum specimens. According to the theory of antigen and antibody, common background proteins would be deleted. The remaining non-binding proteins were analyzed by capillary high-performance liquid chromatography and LTQ-MASS. Nonparametric statistical analysis was used to detect the correlation each differentially expressed protein. RESULTS: The result of HPLC and LTQ-MASS in 30 specimens of patients revealed 4 peaks of differentially expressed proteins including JTR (positive in 18 specimens, 60%), HP( positive in 11 specimens, 36.7%), HPX (positive in 10 specimens, 33.3%), APO (positive in 8 specimens, 26.7%). There were positive correlations between these 4 proteins (the correlation between TTR and HP, HPX, APO is r = 0.480, 0.577, 0.492; the correlation between HP and TTR, HPX, APO is r = 0.480, 0.783, 0.636; the correlation between HPX and TTR, HP, APO is r = 0.577, 0.783, 0.853; the correlation between APO and TTR, HP, HPX is r = 0.492, 0.636, 0.853; P < 0.05). In group of macular hole, TTR was positive expressed in 7 specimens while other differential proteins were low expression. In group of choroidal neovascular, TTR and HP were positive expressed in 6 specimens while HPX was significantly high in 5 specimens. In other two groups, the expression of 4 differential proteins was rather low. CONCLUSIONS: Screening molecular biomarkers by serum-based proteomics can efficiently exclude common proteins and find differential proteins correlated with pathological myopia. These differential proteins may become molecular biomarkers of pathological myopia in the future.

Vitreous proteomic analysis of proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is the most common cause of anatomic failure in retinal detachment surgery. To understand the molecular mechanisms, vitreous proteomes of patients with PVR were investigated by two-dimensional-nano-liquid chromatography coupled with tandem mass spectrometry. Vitreous samples of moderate PVR (grade B), and severe PVR (grade C or D) were aspirated during pars plana vitrectomy before infusion. In the current study, 129, 97 and 137 proteins were identified in vitreous of normal control, moderate and severe PVR, respectively. In PVR vitreous samples, complement components, serine proteinase inhibitors, and extracellular proteins were up-regulated or appeared, while normal cytoskeleton and metabolism proteins were down-regulated or disappeared. It was noteworthy that the proteins involved in transcription and translation regulation increased in vitreous with PVR. Among 102 PVR-specific proteins, kininogen 1 was specifically detected in both vitreous and the corresponding serum. Therefore, it can be concluded that PVR is a complicated pathology process with great amount of proteins involved in metabolism dysfunction, immune reactions, and cytoskeleton remolding. Kininogen 1 may be a candidate biomarker of PVR. Further investigations of these special proteins will provide additional targets for treatment or prevention of ocular proliferative diseases.