Monomeric CXCL12-Engineered Adipose-Derived Stem Cells Transplantation for the Treatment of Ischemic Stroke.

Adipose-derived stem cells (ASCs) possess therapeutic potential for ischemic brain injury, and the chemokine CXCL12 has been shown to enhance their functional properties. However, the cumulative effects of ASCs when combined with various structures of CXCL12 on ischemic stroke and its underlying molecular mechanisms remain unclear. In this study, we genetically engineered mouse adipose-derived ASCs with CXCL12 variants and transplanted them to the infarct region in a mice transient middle cerebral artery occlusion (tMCAO) model of stroke. We subsequently compared the post-ischemic stroke efficacy of ASC-mCXCL12 with ASC-dCXCL12, ASC-wtCXCL12, and unmodified ASCs. Neurobehavior recovery was assessed using modified neurological severity scores, the hanging wire test, and the elevated body swing test. Changes at the tissue level were evaluated through cresyl violet and immunofluorescent staining, while molecular level alterations were examined via Western blot and real-time PCR. The results of the modified neurological severity score and cresyl violet staining indicated that both ASC-mCXCL12 and ASC-dCXCL12 treatment enhanced neurobehavioral recovery and mitigated brain atrophy at the third and fifth weeks post-tMCAO. Additionally, we observed that ASC-mCXCL12 and ASC-dCXCL12 promoted angiogenesis and neurogenesis, accompanied by an increased expression of bFGF and VEGF in the peri-infarct area of the brain. Notably, in the third week after tMCAO, the ASC-mCXCL12 exhibited superior outcomes compared to ASC-dCXCL12. However, when treated with the CXCR4 antagonist AMD3100, the beneficial effects of ASC-mCXCL12 were reversed. The AMD3100-treated group demonstrated worsened neurological function, aggravated edema volume, and brain atrophy. This outcome is likely attributed to the interaction of monomeric CXCL12 with CXCR4, which regulates the recruitment of bFGF and VEGF. This study introduces an innovative approach to enhance the therapeutic potential of ASCs in treating ischemic stroke by genetically engineering them with the monomeric structure of CXCL12.

Engineered exosomes mediated targeted delivery of neuroprotective peptide NR2B9c for the treatment of traumatic brain injury.

Neuroprotection is one of the core treatment strategies for brain injuries including traumatic brain injury (TBI). NR2B9c is a promising neuroprotective peptide but its clinical translation is limited because of poor brain penetrability. Exosomes are naturally occurring nanovesicles having therapeutic potential for TBI as well as an efficient drug delivery carrier to the brain. Here, we engineered exosomes with neuron targeting peptide rabies virus glycoprotein (RVG29) via bio-orthogonal click chemistry technique and loaded it with NR2B9c, developing RVG-ExoNR2B9c. RVG29 conjugated exosome had higher neuron targeting efficiency compared to naïve exosomes both in vivo and in vitro. RVG-ExoNR2B9c had great cytoprotective effect against oxygen glucose deprived Neuro2a cells. Intravenous administration of RVG-ExoNR2B9c significantly improved behavioral outcomes and reduced the lesion volume after TBI injury in a mice controlled cortical impact model. Due to their multifunctionality and significant efficacy, we anticipate that RVG-ExoNR2B9c have the potential to be translated both as therapeutic agent as well as cargo delivery system to the brain for the treatment of TBI.

Bio-clickable, small extracellular vesicles-COCKTAIL therapy for ischemic stroke.

Delivering large therapeutic molecules via the blood-brain barrier to treat ischemic stroke remains challenging. NR2B9c is a potent neuroprotective peptide but it's safe and targeted delivery to the brain requires an efficient, natural, and non-immunogenic delivery technique. Small extracellular vesicles (sEVs) have shown great potential as a non-immunogenic, natural cargo delivery system; however, tailoring of its inefficient brain targeting is desired. Here, we coupled rabies virus glycoprotein 29 with sEVs surface via bio-orthogonal click chemistry reactions, followed by loading of NR2B9c, ultimately generating stroke-specific therapeutic COCKTAIL (sEVs-COCKTAIL). Primary neurons and Neuro-2a cells were cultured for in vitro and transient middle cerebral artery occlusion model was used for in vivo studies to evaluate neuron targeting and anti-ischemic stroke potential of the sEVs-COCKTAIL. Bio-clickable sEVs were selectively taken up by neurons but not glial cells. In the in vitro ischemic stroke model of oxygen-glucose deprivation, the sEVs-COCKTAIL exhibited remarkable potential against reactive oxygen species and cellular apoptosis. In vivo studies further demonstrated the brain targeting and increased half-life of bio-clickable sEVs, delivering NR2B9c to the ischemic brain and reducing stroke injury. Treatment with the sEVs-COCKTAIL significantly increased behavioral recovery and reduced neuronal apoptosis after transient middle cerebral artery occlusion. NR2B9c was delivered to neurons binding to post-synaptic density protein-95, inhibiting N-methyl-d-Aspartate receptor-mediated over production of oxidative stress and mitigating protein B-cell lymphoma 2 and P38 proteins expression. Our results provide an efficient and biocompatible approach to a targeted delivery system, which is a promising modality for stroke therapy.

Progranulin released from microglial lysosomes reduces neuronal ferroptosis after cerebral ischemia in mice.

The cellular redox state is essential for inhibiting ferroptosis. Progranulin (PGRN) plays an important role in maintaining the cellular redox state after ischemic brain injury. However, the effect of PGRN on ferroptosis and its underlying mechanism after cerebral ischemia remains unclear. This study assesses whether PGRN affects ferroptosis and explores its mechanism of action on ferroptosis after cerebral ischemia. We found endogenous PGRN expression in microglia increased on day 3 after ischemia. In addition, PGRN agonists chloroquine and trehalose upregulated PGRN expression, reduced brain infarct volume, and improved neurobehavioral outcomes after cerebral ischemia compared to controls (p < 0.05). Moreover, PGRN upregulation attenuated ferroptosis by decreasing malondialdehyde and increasing Gpx4, Nrf2, and Slc7a11 expression and glutathione content (p < 0.05). Furthermore, chloroquine induced microglial lysosome PGRN release, which was associated with increased neuron survival. Our results indicate that PGRN derived from microglial lysosomes effectively inhibits ferroptosis during ischemic brain injury, identifying it as a promising target for ischemic stroke therapy.

Is TLE1 Expression Limited to Synovial Sarcoma? Our Experience at Shifa International Hospital, Pakistan.

INTRODUCTION: Synovial sarcoma (SS) accounts for 10-15 percent of adult soft tissue sarcomas. Transducin-like enhancer of split 1 (TLE1), a transcriptional repressor, is essential in hematopoiesis, neuronal differentiation, and terminal epithelial differentiation. TLE1 proteins inhibit Wnt signaling and other cell fate determination signals, and so have an established role in repressing differentiation. TLE1 has recently been shown to be a highly sensitive and relatively specific marker of SS. MATERIALS AND METHODS: Study design is retrospective, descriptive. A total of 25 cases of SS and 28 of soft tissue lesions were retrieved from the record. TLE1 (clone 1F5) expression was evaluated and scored as negative (<5% of cells positive), 1+ (5-25% of cells positive), 2+ (26-50% of cells positive), or 3+ (>50 % of cells positive). RESULT: Twenty-four out of twenty-five (96%) cases of SS showed 3+ TLE1 expression. One (4%) case of poorly differentiated SS showed 2+ positivity. 3+ TLE1 positivity was seen in one (100%) case each of infantile fibrosarcoma and low-grade fibromyxoid sarcoma, while two cases (100%) of schwannoma also showed 3+ positivity. All cases of solitary fibrous tumor) (n=2), clear cell sarcoma of tendons and aponeurosis (n=2), embryonal rhabdomyosarcoma (n=1), and de-differentiated liposarcoma (n=2) showed 2+ positivity. 1+ positivity was seen in alveolar soft part sarcoma (n=2), Ewing's sarcoma (n=4), undifferentiated pleomorphic sarcoma (n=1), myxoid liposarcoma (n=1) and malignant peripheral nerve sheath tumor (n=1). TLE1 was negative in all cases of chordomas (n=2), lipomas (n=2), nodular fasciitis (n=2), malignant perivascular epithelioid cell tumor (n=1) and dermatofibrosarcoma protuberans (n=1). CONCLUSION: TLE1 may be a reliable immunostain for diagnosing SS, but its expression is not limited to SS. Its expression should be interpreted in the light of morphological features and a panel of antibodies.

Associations of Most Prevalent Risk Factors with Lung Cancer and Their Impact on Survival Length.

Lung cancer is one of the most common malignancies in the world. Its incidence and mortality rates are on the rise in Pakistan. However, epidemiological studies to identify common lung cancer determinants in the Pakistani population have been limited. In this study, data of 440 cases and 323 controls were collected from different hospitals in Peshawar and Islamabad, along with information about socio-demographic factors including age, sex and smoking. Univariate and multi-factorial analyses of socio-demographic factors in association with each other were also performed. Overall survival analysis highlighted that, out of 440 patients in the lung cancer dataset, 204 people were uncensored with a median survival time of 13 months (95% CI=12-18). There were 41 femaleand 399 male patients. Differences were observed between length of survival in the males and females (χ12 = 6.1; p-value = 0.01). Gender was observed to be significantly related to survival (p-value< 0.01), with better survival in females (hazard ratio=2). Cox regression was extended to adjust for the covariate age (z = 2.5; p-value = 0.02). Survival analysis was also performed on the basis of smoking groups (current smokers, former smokers and never smoked individuals) and smoking duration (smoking duration>10 years, <10 years and never smoked). Smoking duration was significantly associated with survival (p-value < 0.01), with better survival in never smokers in comparison to both smoking for greater or less than 10 years. Strong associations were observed for smoking group with duration greater than 10 years, OR=6.1(3.9-9.5) on univariate and multi-factorial analysis OR=11.3(CI=6.8-19.3).

Association of the CYP17-34T/C Polymorphism with Pancreatic Cancer Risk.

Pancreatic cancer is a leading cause of fatality worldwide. Several population studies have been conducted on genetic diagnosis of pancreatic cancer but the results from epidemiologic studies are very limited. CYP17A gene has a role in disease formation but its influence on pancreatic cancer is unclear. A polymorphism in the 5'UTR promoter region of CYP17A1-34T/C (A1/A2) has been associated with multiple cancers. The aim of the current study was to assess associations of this polymorphism and socio-demographic risk factors with pancreatic cancer. A total of 255 and 320 controls were enrolled in the study, and were genetically analyzed through PCR-RFLP. Statistical analysis was conducted with observed genotype frequencies and odds ratios (ORs) and 95% CIs were estimated using unconditional logistic regression. The impact of socio-demographic factors was accessed through Kaplen-Meir analysis. According to our results, the A2/A2 genotype was significantly associated with pancreatic cancer (OR=2.1, 95%CI = 1.3-3.5). Gender female (OR=2.6, 95%CI=1.8-3.7), age group 80s/80+ years (OR=2.2, 95% CI=1.2-4), smoking both former (OR=4.6, 95% CIs=2.5-8.8) and current (OR=3.6, 95% CI=2-6.7), and family history (OR=7.1; 95%CI = 4.6-11.4) were also found associated with increased risk. Current study suggests that along with established risk factors for pancreatic cancer CYP17A1-34T/C may play a role. However, on the basis of small sample size the argument cannot be fully endorsed and larger scale studies are recommended.

Immunoglobulin M Nephropathy in a Patient with Wilson's Disease.

Immunoglobulin M nephropathy (IgMN) is characterized by the deposition of immunoglobulin M in a dominant distribution in the renal glomeruli. Primary immunoglobulin M nephropathy is diagnosed after consistent light microscopy (LM), immunofluorescence (IF), electron microscopy (EM) results, and exclusion of known systemic disorders causing immunoglobulin M deposition in the glomeruli. The secondary disease has been reported with a few conditions though it has never been reported with any primary disease of the liver. We report the case of an adolescent male patient who presented with nausea, vomiting, diarrhea, and worsening anasarca. He was found to have nephrotic-range proteinuria that did not respond to conventional corticosteroid treatment. He was subjected to a renal biopsy which revealed a diagnosis of immunoglobulin M nephropathy. His liver function tests were deranged and an ultrasound scan of the abdomen revealed a coarse irregular liver. Workup revealed elevated urine copper excretion and a low ceruloplasmin level. He was diagnosed as a case of Wilson's disease and started on penicillamine and pyridoxine. He was also started on intravenous cyclophosphamide for the corticosteroid-resistant nephrotic syndrome to which he responded remarkably well. His edema settled, proteinuria resolved, and liver functions normalized. Currently, he is in remission and enjoying good health. To the best of our knowledge, we report the first known association between IgM nephropathy and Wilson's disease. It is presently not clear if causation can necessarily be established. This may be the result of defective IgM clearance by the liver or an altered metabolism of the antibody or immune complexes, as with hepatic-associated immunoglobulin M (IgM) nephropathy. Further studies are needed to elucidate the exact mechanism of this disease.