Macrophage polarization impacts tunneling nanotube formation and intercellular organelle trafficking.

Tunneling nanotubes (TNTs) are cellular extensions enabling cytosol-to-cytosol intercellular interaction between numerous cell types including macrophages. Previous studies of hematopoietic stem and progenitor cell (HSPC) transplantation for the lysosomal storage disorder cystinosis have shown that HSPC-derived macrophages form TNTs to deliver cystinosin-bearing lysosomes to cystinotic cells, leading to tissue preservation. Here, we explored if macrophage polarization to either proinflammatory M1-like M(LPS/IFNγ) or anti-inflammatory M2-like M(IL-4/IL-10) affected TNT-like protrusion formation, intercellular transport and, ultimately, the efficacy of cystinosis prevention. We designed new automated image processing algorithms used to demonstrate that LPS/IFNγ polarization decreased bone marrow-derived macrophages (BMDMs) formation of protrusions, some of which displayed characteristics of TNTs, including cytoskeletal structure, 3D morphology and size. In contrast, co-culture of macrophages with cystinotic fibroblasts yielded more frequent and larger protrusions, as well as increased lysosomal and mitochondrial intercellular trafficking to the diseased fibroblasts. Unexpectedly, we observed normal protrusion formation and therapeutic efficacy following disruption of anti-inflammatory IL-4/IL-10 polarization in vivo by transplantation of HSPCs isolated from the Rac2-/- mouse model. Altogether, we developed unbiased image quantification systems that probe mechanistic aspects of TNT formation and function in vitro, while HSPC transplantation into cystinotic mice provides a complex in vivo disease model. While the differences between polarization cell culture and mouse models exemplify the oversimplicity of in vitro cytokine treatment, they simultaneously demonstrate the utility of our co-culture model which recapitulates the in vivo phenomenon of diseased cystinotic cells stimulating thicker TNT formation and intercellular trafficking from macrophages. Ultimately, we can use both approaches to expand the utility of TNT-like protrusions as a delivery system for regenerative medicine

Assessment of contamination on sterilised dental burs after being subjected to various pre-cleaning methods

OBJECTIVE: To determine the effectiveness of different pre-cleaning methods by determining frequency and site of contamination on the sterilised dental burs using Phloxine B dye. METHODS: The in-vitro experimental study was conducted from June to August 2017 at dental clinics of Aga Khan University Hospital Karachi. Diamond dental burs were selected and divided into two control and four test groups. The two control groups were classified as Negative (new burs) and Positive (used contaminated). The four test groups were classified as Manual (Group-1), Ultrasonic (Group-2), Manual + Enzyme (Group-3) and Manual + Ultrasonic (Group-4). Phloxine B dye was used to determine the contamination. The images of the burs were taken and enlarged at 15X before subjected to visual assessment. Association between contamination and pre-cleaning methods were determined. Data was analysed using SPSS version 22. RESULTS: A total of 210 burs were selected for the study which were divided in 6 groups of 35(16.66%) each. One (2.8%) bur in negative control group and all burs in positive control group showed contamination. In test groups, 27(77.1%), 29(82.8%), 27(77.1%) and 24(68.5%) burs showed contamination in groups 1, 2, 3 and 4, respectively. There was no association between type of pre-cleaning method with the frequency of contamination (p =0.57). The head of bur was the most frequently contaminated site (p \u3c 0.003). CONCLUSIONS: None of the pre-cleaning method was found to be effective. Head of bur was the most frequently contaminated site

Visual and microscopic evaluation of the surface alterations In the protaper files after single clinical use

BACKGROUND: Different studies have been conducted in which defects of Ni-Ti files were reported after multiple usages but limited data is available regarding the defects in the rotary Ni-Ti files subjected to single clinical use. The objective of this study was to determine the frequency of surface defects caused by fatigue in the rotary ProTaper files after single clinical use assessed with visual and microscopic examination methods. METHODS: A cross-sectional study was conducted in the dental clinics of The Aga Khan University Hospital, Karachi, Pakistan. A total of 189 ProTaper Ni-Ti files (after single clinical use in multi-rooted molars) were analysed visually and then under stereomicroscope at 10Χ magnification for surface defects (straightening, denting, bending, twisting, pitting and change in length). Chi Square test was used to determine association between type of file and type of defect. Spearman\u27s correlation test was used for determination of correlation between visual and microscopic examinations at 0.05 level of significance. RESULTS: 19% of files showed straightening on visual assessment as compared to 66.1% under microscopic examination. There was a statistically significant association between the file type and the straightening of file (p-value ≤0.001). A weak correlation existed between visual and microscopic examination for all the defects, except for the change in length. CONCLUSIONS: The defects of ProTapers files are best detected by the microscopic examination. Straightening is the most common defect observed visually and microscopically. The first shaping and first finishing files underwent significantly more surface defects than the rest of the rotary files in the series

Most effective method for the management of physiologic gingival hyperpigmentation: A systematic review and meta-analysis

MATERIALS AND METHODS: A systematic review and meta-analysis were done (1919 to October 2018) using PubMed, CINHAL, Dental and Oral Science, and manual searches. Twenty-five articles were finally reviewed. Only human clinical trials were considered with physiological gingival pigmentation treated with different depigmentation methods and compared with surgical stripping. The outcome was the achievement of gingival depigmentation and its recurrence. RevMan software was used for data analysis. RESULTS: Of 26,132 articles, 25 met the inclusion criteria. Seventeen were randomized control trials and 8 were nonrandomized control trials. Most of the studies were on laser. The control group was scalpel surgery. Majority of studies showed no difference in compared treatment modality. A meta-analysis compared laser ablation with surgical stripping revealed a nonsignificance difference regarding recurrence (P = 0.75) and depigmentation (P = 0.23) and a statistically significant difference regarding postoperative pain favoring laser ablation (P ≤ 0.05). CONCLUSIONS: Surgical stripping has been the conventional treatment of choice, but our review showed that new techniques are equally effective or even better. Laser especially diode laser was the most frequently used technique and showed better esthetic outcomes, less pain, faster healing, and patients\u27 preference and satisfaction after treatment. However, laser showed more regimentation at 6-month evaluation. More good quality randomized controlled trials with different depigmentation methods are needed to draw strong conclusions

Cellular and Biochemical Underpinnings of Stem Cell Gene Therapies for Cystinosis

Cystinosis is an early onset multisystemic lysosomal storage disorder characterized by deleterious CTNS gene mutations causing defective export and crystallization of amino acid dimer cysteine within lysosomes. Previous studies found that wild-type hematopoietic stem and progenitor cell (HSPC) transplantation into cystinotic (Ctns-/-) mice prevents disease progression via HSPC-derived macrophage-mediated TNT delivery of functional Ctns-carrying lysosomes to diseased cells. While Phase I/II clinical trials for ex vivo lentivirally gene-corrected autologous HSPC transplantation is ongoing at UC San Diego, this approach does not yet extend to 40% of patients harboring homozygous 57 kilobase pair mutations eliminating both CTNS and neighboring pentose phosphate pathway SHPK genes. With roles in both metabolism and macrophage polarization, understanding the clinical relevance of SHPK to our macrophage-based gene therapy is essential to elucidate if SHPK-deficient patients are likely to benefit from the therapy. In this pursuit, we examined the role of polarization conditions mediating macrophage-derived TNT intracellular trafficking in robust in vitro and in vivo systems. We generated novel Shpk knockout (Shpk-/-) mice models which upheld mild metabolic phenotypes as a consequence of isolated Shpk-deficiency. Furthermore, we found that Shpk-/-/Ctns+/+ HSPC transplantation into lethally-irradiated cystinotic Ctns-/-/Shpk+/+ mice resulted in normal macrophage tissue integration, restored Ctns mRNA expression, reduced cystine content, and improved renal function, inducing widespread disease rescue. Ultimately, these findings provide insight into the pathogenic role of SHPK in cystinosis, validate the utility of HSPC transplantations as gene-modifying therapies for disorders of similar cellular/molecular underpinnings, and confirms eligibility of patients with SHPK-deficiency for cystinosis clinical trials

Macrophage polarization impacts tunneling nanotube formation and intercellular organelle trafficking.

Tunneling nanotubes (TNTs) are cellular extensions enabling cytosol-to-cytosol intercellular interaction between numerous cell types including macrophages. Previous studies of hematopoietic stem and progenitor cell (HSPC) transplantation for the lysosomal storage disorder cystinosis have shown that HSPC-derived macrophages form TNTs to deliver cystinosin-bearing lysosomes to cystinotic cells, leading to tissue preservation. Here, we explored if macrophage polarization to either proinflammatory M1-like M(LPS/IFNγ) or anti-inflammatory M2-like M(IL-4/IL-10) affected TNT-like protrusion formation, intercellular transport and, ultimately, the efficacy of cystinosis prevention. We designed new automated image processing algorithms used to demonstrate that LPS/IFNγ polarization decreased bone marrow-derived macrophages (BMDMs) formation of protrusions, some of which displayed characteristics of TNTs, including cytoskeletal structure, 3D morphology and size. In contrast, co-culture of macrophages with cystinotic fibroblasts yielded more frequent and larger protrusions, as well as increased lysosomal and mitochondrial intercellular trafficking to the diseased fibroblasts. Unexpectedly, we observed normal protrusion formation and therapeutic efficacy following disruption of anti-inflammatory IL-4/IL-10 polarization in vivo by transplantation of HSPCs isolated from the Rac2-/- mouse model. Altogether, we developed unbiased image quantification systems that probe mechanistic aspects of TNT formation and function in vitro, while HSPC transplantation into cystinotic mice provides a complex in vivo disease model. While the differences between polarization cell culture and mouse models exemplify the oversimplicity of in vitro cytokine treatment, they simultaneously demonstrate the utility of our co-culture model which recapitulates the in vivo phenomenon of diseased cystinotic cells stimulating thicker TNT formation and intercellular trafficking from macrophages. Ultimately, we can use both approaches to expand the utility of TNT-like protrusions as a delivery system for regenerative medicine

Deficiency of the sedoheptulose kinase (Shpk) does not alter the ability of hematopoietic stem cells to rescue cystinosis in the mouse model.

Cystinosis is an autosomal recessive lysosomal storage disorder caused by mutations in the CTNS gene encoding the lysosomal cystine transporter, cystinosin, and leading to multi-organ degeneration including kidney failure. A clinical trial for cystinosis is ongoing to test the safety and efficacy of transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) ex vivo gene-modified to introduce functional CTNS cDNA. Preclinical studies in Ctns-/- mice previously showed that a single HSPC transplantation led to significant tissue cystine decrease and long-term tissue preservation. The main mechanism of action involves the differentiation of the transplanted HSPCs into macrophages within tissues and transfer of cystinosin-bearing lysosomes to the diseased cells via tunneling nanotubes. However, a major concern was that the most common cystinosis-causing mutation in humans is a 57-kb deletion that eliminates not only CTNS but also the adjacent sedopheptulose kinase SHPK/CARKL gene encoding a metabolic enzyme that influences macrophage polarization. Here, we investigated if absence of Shpk could negatively impact the efficiency of transplanted HSPCs to differentiate into macrophages within tissues and then to prevent cystinosis rescue. We generated Shpk knockout mouse models and detected a phenotype consisting of perturbations in the pentose phosphate pathway (PPP), the metabolic shunt regulated by SHPK. Shpk-/- mice also recapitulated the urinary excretion of sedoheptulose and erythritol found in cystinosis patients homozygous for the 57-kb deletion. Transplantation of Shpk-/--HSPCs into Ctns-/- mice resulted in significant reduction in tissue cystine load and restoration of Ctns expression, as well as improved kidney architecture comparable to WT-HSPC recipients. Altogether, these data demonstrate that absence of SHPK does not alter the ability of HSPCs to rescue cystinosis, and then patients homozygous for the 57-kb deletion should benefit from ex vivo gene therapy and can be enrolled in the ongoing clinical trial. However, because of the limits inherent to animal models, outcomes of this patient population will be carefully compared to the other enrolled subjects