Single fibers of skeletal muscle as a novel graft for cell transplantation to the heart

OBJECTIVE: Skeletal myoblast transplantation is a promising alternative to treat heart failure. A single fiber, the minimal functional unit of skeletal muscle, retains skeletal myoblasts beneath the basal lamina. When surrounding muscle is injured, myoblasts migrate from the fiber into the damaged area to regenerate muscle. We hypothesized that such isolated fibers could be used as an efficient vehicle to deliver myoblasts into damaged myocardium, resulting in improved cardiac function. METHODS: Living single fibers of rat skeletal muscle were isolated, and their behavior was characterized in vitro. Single fibers were injected into the myocardium (at 4 sites, each receiving a single fiber) of rats in 2 models of heart failure induced either by means of doxorubicin administration or left coronary artery occlusion. RESULTS: Skeletal myoblasts dissociated from an isolated single fiber, proliferated, and differentiated into multinucleated myotubes in vitro. Within 3 days after grafting in vivo, original fibers provided putative myoblasts and disappeared. At 4 weeks, discrete loci consisting of several multinucleated myotubes were observed. Furthermore, single-fiber transplantation significantly improved cardiac function compared with the control treatment in either doxorubicin-treated hearts (maximum dP/dt, 4013.9 +/- 96.1 vs 3603.1 +/- 102.3 mm Hg/s; minimum dP/dt, -2313.7 +/- 75.1 vs. -2057.1 +/- 52.4 mm Hg/s) or ischemic hearts (maximum dP/dt, 3905.6 +/- 103.0 vs 3572.6 +/- 109.7 mm Hg/s; minimum dP/dt, -2336.1 +/- 69.7 vs -2106.4 +/- 74.2 mm Hg/s). CONCLUSION: Single-fiber transplantation acts as a vehicle for delivering putative skeletal myoblasts that appear to differentiate into myotubes within the myocardium. This was associated with improved function of failing hearts, suggesting its efficacy as a novel graft for cellular cardiomyoplasty.     

Progesterone improves acute recovery after traumatic brain injury in the aged rat

Recent evidence has demonstrated that treatment with progesterone can attenuate many of the pathophysiological events following traumatic brain injury (TBI) in young adult rats, but this effect has not been investigated in aged animals. In this study, 20-month-old male Fischer 344 rats with bilateral contusions of the frontal cortex (n = 4 per group) or sham operations received 8, 16, or 32 mg/kg of progesterone or vehicle. Locomotor activity was measured at 72 h to assess behavioral recovery. Brain tissue was harvested at 24, 48, and 72 h, and Western blotting was performed for inflammatory and apoptotic factors. Edema was assessed at 48 h by measuring brain water content. Injured animals treated with 8 and 16 mg/kg progesterone showed decreased expression of COX-2, IL-6, and NFkappaB at all time points, indicating a reduction in the acute inflammatory process compared to vehicle. The 16 mg/kg group also showed reduced apoptosis at all time points as well as decreased edema and improved locomotor outcomes. Thus, in aged male rats, treatment with 16 mg/kg progesterone improves short-term motor recovery and attenuates edema, secondary inflammation, and cell death after TBI.     

ALT-946 and aminoguanidine,inhibitors of advanced glycation,improve severe nephropathy in the diabetic transgenic (mREN-2)27 rat

The severe diabetic nephropathy that develops in the hypertensive transgenic (mRen-2)27 rat with streptozotocin (STZ) diabetes has previously been considered angiotensin II-dependent. Because metabolic pathways are also activated in the diabetic kidney, the present study aimed to determine whether renoprotection could be afforded with inhibitors of advanced glycation end products (AGEs), ALT-946, and aminoguanidine (AG). At 6 weeks of age, nondiabetic control and STZ diabetic Ren-2 rats were randomized to receive vehicle, ALT-946 (1 g/l), or AG (1 g/l) and were studied for 12 weeks. Systolic blood pressure was unchanged with diabetes, ALT-946, or AG. Both kidney weight and glomerular filtration rate were increased with diabetes and unchanged with ALT-946 or AG. ALT-946 and AG equally ameliorated glomerulosclerosis and medullary pathology; however, ALT-946 did reduce cortical tubular degeneration to a greater extent than AG. Albumin excretion rate, which was elevated with diabetes, was reduced with ALT-946 but not AG. AGE immunolabeling was increased in glomeruli and reduced with ALT-946 and AG. These findings indicate that even in the context of renal injury presumed to be primarily blood pressure- and/or angiotensin II-dependent, approaches that interfere with metabolic pathways such as inhibitors of AGE formation can confer renal protection in experimental diabetes.     

Sulfonylurea-sensitive K(+) transport is involved in Cl(-) secretion and cyst trowth by cultured ADPKD cells

Transepithelial chloride and fluid secretion by many types of epithelia involves activation of a conductive K(+) pathway that serves to support the electrochemical driving force for Cl(-) secretion. This study sought to determine if such a pathway is involved in Cl(-) and fluid secretion by the cystic epithelia in autosomal dominant polycystic kidney disease (ADPKD). Primary cultures of cells derived from the cysts of patients with ADPKD were used. Confluent monolayers of these cells, mounted in Ussing chambers, were stimulated to secrete Cl(-) by application of the adenylyl cyclase agonist, forskolin. The effects of various K(+) channel blockers on the increase in short-circuit current (I(sc)) generated by active Cl(-) secretion were determined. Charybdotoxin, an inhibitor of Ca(2+)-sensitive K(+) channels exerted no effect. Similarly, the chromanole 293B, an inhibitor of cAMP-induced K(+) conductance, exerted no effect on cAMP-dependent anion secretion. Glibenclamide, an inhibitor of ATP-sensitive K(+) channels and the cystic fibrosis transmembrane conductance regulator (CFTR), modestly inhibited the forskolin-stimulated current when applied to the apical surface of the monolayers, suggesting a relatively weak effect on CFTR. Basolateral application of glibenclamide inhibited I(sc) to a greater extent. This latter effect may be due to inhibition of a K(+)-conductive transport step. Glibenclamide exerted little effect on the I(sc) of nonstimulated monolayers. Cyst growth in ADPKD is driven by cell proliferation and Cl(-) and fluid secretion. The effect of glibenclamide on the growth of cysts formed within a collagen gel by cultured ADPKD cells was tested. Addition of glibenclamide to the media bathing the cysts inhibited their growth. Glibenclamide also blocked the formation of cysts when it was added to the media at the time the cells were seeded within the collagen gel. Glibenclamide was also found to inhibit the proliferation of ADPKD cells. RT-PCR analysis demonstrated that the ATP-sensitive K(+) channel, K(ir) 6.2, is expressed in cultured ADPKD cells and in normal human kidney. These results suggest that ATP-sensitive K(+) channel blockers should be investigated as possible therapeutic agents to inhibit cyst growth in ADPKD.     

Posterior Surgical Treatment of Cervical Spondylotic Myelopathy: Review Article

Background

Cervical spondylosis is now recognised as the leading cause of myelopathy and spinal cord dysfunction worldwide. Chronic spinal cord compression results in chronic inflammation, cellular apoptosis, and microvacular insufficiency, which are thought to the biologic basis for cervical spondylotic myelopathy (CSM).

Questions/Purposes

Our purpose was to address the key principles of CSM, including natural history and presentation, pathogenesis, optimal surgical approach, results and complication rates of posterior surgical approaches for CSM so that the rationale for addressing CSM by a posterior approach can be fully understood.

Methods

We conducted a systematic search of PubMed/MEDLINE and the Cochrane Collaboration Library for literature published through February 2014 to identify articles that evaluated CSM and its management. Reasons for exclusion included patients with ossification of the posterior longitudinal ligament (OPLL), patients with degenerative disc disease without CSM, and patients with spine tumor, trauma and infection. Meeting abstracts/proceedings, white articles and editorials were additionally excluded.

Results

The search strategy yielded 1,292 articles, which was reduced to 52 articles, after our exclusion criteria were introduced. CSM is considered to be a surgical disorder due to its progressive nature. There is currently no consensus in the literature whether multilevel spondylotic compression is best treated via an anterior or posterior surgical approach.

Conclusion

Multilevel CSM may be safely and effectively treated using a posterior approach, either by laminoplasty or with a laminectomy and fusion technique.

Electronic supplementary material

The online version of this article (doi:10.1007/s11420-014-9425-5) contains supplementary material, which is available to authorized users.     

Matrix metalloproteinase-13 influences ERK signalling in articular rabbit chondrocytes

OBJECTIVE: Matrix metalloproteinase-13 (MMP-13) is an extracellular MMP that cleaves type II collagen, the major protein component of cartilage, with high specificity and has been implicated in the pathology of osteoarthritis. The present study aimed to characterize the binding and internalization kinetics of MMP-13 in normal rabbit chondrocytes and whether MMP-13 affected cell signalling. METHODS: Rabbit chondrocytes were used in [125I]-MMP-13 binding assays to investigate the MMP-13 binding kinetics and Western analysis allowed for the assessment of intracellular signalling cascades. RESULTS: Rabbit chondrocytes were found to express the cartilage-specific genes aggrecan and type II collagen throughout their in vitro culture period. Appreciable specific cell-association of [125I]-MMP-13 was detected after 10 min of exposure to the ligand and equilibrium was obtained after 2 h. Binding of [125I]-MMP-13 to chondrocytes was specific and approached saturation at 75 nM. Internalization of MMP-13 was evident after 20 min, reached a maximum at 30 min and had returned to baseline by 90 min. Addition of receptor-associated protein (RAP) inhibited the internalization of MMP-13 indicating a likely role for low-density lipoprotein receptor-related protein-1 (LRP1) in this process. Interestingly the presence of MMP-13 induced phosphorylation of the extracellular signal-regulated kinase 1/2 (ERK1/2) protein showing that there is initiation of a signalling process in response to MMP-13 being bound and internalized by rabbit chondrocytes. However, this activation does not involve the MMP-13 internalization receptor LRP1. CONCLUSION: These studies demonstrate and characterize the MMP-13 binding and internalization system in rabbit chondrocytes and indicate that MMP-13 may regulate the phenotype of the chondrocytes through this receptor system.     

Does the false-negative rate for 1 or 2 negative sentinel nodes after neo-adjuvant chemotherapy translate into a high local recurrence rate?

Prospective trials demonstrate that sentinel node (SN) biopsy after neo-adjuvant chemotherapy (NACT) has a significant false-negative rate (FNR) when only 1 or 2 SNs are removed. It is unknown whether this increased FNR correlates with an elevated risk of recurrence. Tumor Registry data at an NCI-Designated Comprehensive Cancer Center were reviewed from 2004 to 2018 for patients having a negative SN biopsy after NACT. Among 190 patients with histologically negative nodes after NACT having 1 (n = 42), 2 (n = 46), and ≥3 (n = 102) SNs, axillary recurrences occurred in 7.14%, 0%, and 1.96% (p = 0.09), breast recurrences occurred in 2.38%, 6.52%, and 0.98% (p = 0.12), and distance recurrences occurred in 16.67%, 8.70%, and 7.84% (p = 0.27), respectively. Time to first recurrence did not differ by SN count (p = 0.41). After adjustment for age, race, clinical stage, and receptor status, there were no differences in the rates of axillary (p = 0.26), breast (p = 0.44), or distance recurrence (p = 0.24) by numbers of SNs harvested. Median follow-up was 46.8 months. Despite higher post-NACT FNRs reported in randomized trials for patients having <3 sentinel nodes, recurrence rates were not significantly different for 1 versus 2 versus ≥3 SNs. This suggests that patients having 1 or 2 post-NACT SNs identified may not necessitate axillary dissection.     

Angiotensin-converting enzyme inhibition attenuates renal platelet-derived growth factor gene expression and cell proliferation in subtotal nephrectomy

Cell proliferation, matrix accumulation and cell infiltration are characteristic features of progressive glomerulosclerosis and tubulointerstitial fibrosis. Platelet‐derived growth factor (PDGF), a cytokine which has proliferative, prosclerotic and chemokine properties, has been shown to be upregulated in the rat remnant kidney model. Inhibition of the renin–angiotensin system by angiotensin‐converting enzyme (ACE) inhibitors has a beneficial effect on renal function and morphology, but the effect of ACE inhibition on PDGF gene expression and PDGF‐mediated cellular proliferation in subtotal nephrectomy has not been studied in detail. Twelve rats were subtotally nephrectomized (STNx) and received either the ACE inhibitor perindopril or a placebo for 12 weeks. Five sham‐operated rats served as controls. Subtotal nephrectomy was associated with hypertension, proteinuria, elevated plasma creatinine and increased kidney weight. After 12 weeks, PDGF B‐chain mRNA was significantly upregulated in the glomeruli and tubulointerstitium of subtotally nephrectomized rats. ACE inhibition attenuated PDGF mRNA expression in association with a reduction in tubular and glomerular proliferation, as assessed by staining for proliferating cell nuclear antigen. In the context of the known in vitro and in vivo effects of PDGF, it is postulated that the renoprotective action of ACE inhibitors may be partially related to PDGF‐mediated antiproliferative mechanisms.     

Increased expression of cyclooxygenase and nitric oxide isoforms, and exaggerated sensitivity to prostaglandin E2, in the rat lumbar spinal cord 3 days after L5-L6 spinal nerve ligation

BACKGROUND: Spinal prostaglandins seem to be important in the early pathogenesis of experimental neuropathic pain. Here, the authors investigated changes in the expression of cyclooxygenase and nitric oxide synthase (NOS) isoforms in the lumbar, thoracic, and cervical spinal cord and the pharmacologic sensitivity to spinal prostaglandin E2 (PGE2) after L5-L6 spinal nerve ligation (SNL). METHODS: Male Sprague-Dawley rats, fitted with intrathecal catheters, underwent SNL or sham surgery 3 days before experimentation. Paw withdrawal threshold was monitored for up to 20 days. Immunoblotting, spinal glutamate release, and behavioral testing were examined 3 days after SNL. RESULTS: Allodynia (paw withdrawal threshold < or = 4 g) was evident 1 day after SNL and remained stable for 20 days. Paw withdrawal threshold was unchanged (P > 0.05) from baseline (> 15 g) after sham surgery except for a small but significant decrease on day 20. Cyclooxygenase 2, neuronal NOS, and inducible NOS were significantly increased in the ipsilateral lumbar dorsal horn after SNL. Expression in the contralateral dorsal horn and ventral horns (lumbar segments) or bilaterally (thoracic and cervical segments) was unchanged from sham controls. This was accompanied by a significant decrease in both the EC50 of PGE2-evoked glutamate release and the ED50 of PGE2 on brush-evoked allodynia. Enhanced sensitivity to PGE2 was localized to lumbar segments of SNL animals and attenuated by SC-51322 or S(+)-ibuprofen, but not R(-)-ibuprofen (100 mum). CONCLUSION: The increased expression of cyclooxygense-2, neuronal NOS, and inducible NOS and the enhanced sensitivity to PGE2 in spinal segments affected by SNL support the hypothesis that spinal prostanoids play an early pathogenic role in experimental neuropathic pain.     

Advances in Predicting and Manipulating the Immunogenicity of Biotherapeutics and Vaccines

Therapeutic proteins are vital to the future of human health provision and the survival and profitability of the global pharmaceutical industry. Returns from protein therapeutics are experiencing unprecedented growth: both their number and their economic dividend have increased by an order of magnitude in the last 10 years. The potential immunogenicity of protein therapeutics raises many clinical and safety concerns. Many poorly understood factors relating to both product and host affect immune responses. Available laboratory measurement of immunogenicity is of little utility for predicting the clinical properties of biotherapeutics. Coupled with assay variability and standardization issues, this precludes adequate prediction of the biological or clinical responses of therapeutic proteins, arguing for the utilization of informatic strategies in the analysis and prediction of protein immunogenicity. Currently, many unresolved issues must be addressed and thus circumvented before effective prediction can become routine.