Ana Coelho

Distribution of the High-Affinity Binding Site and Intracellular Target of Botulinum Toxin Type A in the Human Bladder

BACKGROUND Botulinum toxin type A (BoNTA) has been successfully used in the treatment of refractory detrusor overactivity. The toxin is internalized after binding a high-affinity receptor, synaptic vesicle protein 2 (SV2), which is exposed in the cell membrane during the exocytosis process. In the cytoplasm, BoNTA cleaves specific sites of synaptosomal-associated protein 25 (SNAP-25), preventing the assembly of the synaptic fusion complex SNARE and blocking exocytosis. OBJECTIVE In the present work, the distribution of SV2 and SNAP-25 was first investigated in human bladders. The neurochemistry of BoNTA-sensitive structures was then investigated using markers for parasympathetic, sympathetic, and sensory fibers. DESIGN, SETTING, AND PARTICIPANTS Human bladders were obtained from cadaveric organ donors (age range: 19-74 yr). MEASUREMENTS Bladder sections were processed for single or dual immunofluorescence staining with antibodies against SV2, SNAP-25, β-3 tubulin, vesicular acetylcholine transporter, tyrosine hydroxilase, and calcitonin gene-related peptide. RESULTS AND LIMITATIONS SV2 and SNAP-25 immunoreactive fibers were distributed throughout the suburothelium and muscular layer. Double labeling showed extensive colocalization of both proteins in nerve fibers. SV2 is more expressed in parasympathetic fibers than in sympathetic or sensory fibers. No expression was found in urothelial or muscular cells. Because only normal bladders were used, this distribution should be applied with caution to pathologic bladders. CONCLUSIONS SV2 and SNAP-25 colocalize abundantly throughout the urinary bladder. SV2 is more abundant in cholinergic, parasympathetic fibers. These nerves are suggested to be the main target for BoNTA action in the human urinary bladder.

Spread of OnabotulinumtoxinA After Bladder Injection. Experimental Study Using the Distribution of Cleaved SNAP-25 as the Marker of the Toxin Action

BACKGROUND OnabotulinumtoxinA (Onabot/A) has been used to treat detrusor overactivity disorders. The treatment is based on several injections of toxin throughout the bladder wall. However, injection protocols are not well established among clinicians, varying in dose and dilution. OBJECTIVE Study the distribution and neurochemistry of cleaved synaptosome-associated protein of 25 kDa (cSNAP-25) after Onabot/A administration in the guinea pig bladder. In addition, we analyzed which factor, dose or volume, contributes more to the diffusion of the toxin. DESIGN, SETTING, AND PARTICIPANTS Guinea pig bladders were treated with Onabot/A via intramural injection or an instillation. MEASUREMENTS Bladder cryostat sections were processed for single or dual immunohistochemistry staining with antibodies against cSNAP-25, vesicular acetylcholine transporter, tyrosine hydroxylase, and calcitonin gene-related peptide. Different administration methods and doses were analyzed. Statistical analysis was performed using the chi-square test for colocalization studies after multiple injections and the t test for the evaluation of affected fibers after a single injection. RESULTS AND LIMITATIONS cSNAP-25 immunoreactive fibers were abundant throughout the bladder tissue in the mucosa and muscular layer. Double labeling showed that parasympathetic fibers are more affected than sympathetic or sensory. A single Onabot/A injection is more effective if diluted in a higher volume. Onabot/A instillation in the bladder does not cleave SNAP-25 protein. CONCLUSIONS A single Onabot/A injection spreads the neurotoxin activity to the opposite side of the guinea pig bladder. This action is more evident when high saline volumes are used to dissolve Onabot/A. The toxin cleaves the SNAP-25 protein mainly in cholinergic but also in adrenergic and sensory fibers. In contrast with intramural injection, instillation of Onabot/A does not cleave SNAP-25 in nerve fibers.

Effect of OnabotulinumtoxinA on Intramural Parasympathetic Ganglia: An Experimental Study in the Guinea Pig Bladder

PURPOSE We investigated whether onabotulinumtoxinA injected in the bladder would affect preganglionic parasympathetic nerve endings in intramural ganglia. MATERIALS AND METHODS Guinea pig bladders were injected with 5 U of botulinum toxin. At 24 hours bladders were collected and processed for immunohistochemistry using tyrosine hydroxylase, and intact and cleaved SNAP-25. To identify the different populations of affected fibers coursing the ganglia we performed double immunoreactions for cleaved SNAP-25 and VAChT, TH or CGRP. RESULTS VAChT immunoreactive fibers were identified in axons and varicosities of presynaptic to postganglionic parasympathetic neurons. Those fibers were also immunoreactive to SV2 and SNAP-25. The rare CGRP and TH immunoreactive fibers coursing in the ganglia did not express SV2 or SNAP-25. After onabotulinumtoxinA injection the cleaved form of SNAP-25 was abundantly expressed in parasympathetic fibers. CONCLUSIONS Botulinum toxin injection in the bladder wall affects preganglionic parasympathetic nerve terminals. This could contribute to the strong effect of botulinum toxin on bladder smooth muscle activity.

Urinary bladder inflammation induces changes in urothelial nerve growth factor and TRPV1 channels.

The urinary bladder urothelium expresses various receptors and in response to chemical and mechanical stimuli releases mediators, thereby modulating bladder sensory pathways. Transient receptor potential vanilloid 1 (TRPV1) ion channels and nerve growth factor (NGF) in those cells are implicated in this modulatory effect and play a role in sensitizing pain‐related afferent pathways during inflammation. In this study, we investigated the interaction between NGF and TRPV1 channels in urothelial cells.

Intrathecal administration of botulinum toxin type A improves urinary bladder function and reduces pain in rats with cystitis

Botulinum toxin A (Onabot/A) has been shown to have an antinociceptive effect. This might be due to an impairment of sensory nerves not only in the peripheral but also in the central nervous system. In this work, we analysed both systems by studying the effect of intrathecal (i.t.) administration of botulinum toxin A in an animal model of bladder pain and hyperactivity induced by cyclophosphamide (CYP).

Phase II Study of Celecoxib with Cisplatin Plus Etoposide in Extensive-Stage Small Cell Lung Cancer

We performed a phase II trial to test whether a cyclooxygenase (COX-2) inhibitor, celecoxib, added to standard first-line combination chemotherapy (CT) and as maintenance therapy would improve outcomes in extensive-stage (ES) small-cell lung cancer (SCLC). This was a multicenter trial in CT-naive patients with ES-SCLC. They received standard cisplatin and etoposide (EP) up to 6 cycles and celecoxib 400 mg PO bid continuously until disease progression. Primary end points were response rate (RR), time to progression (TTP), and toxicity. Secondary were overall survival (OS) and quality of life. Of 74 expected patients, only 24 were enrolled and the study stopped earlier because of the published safety concerns about celecoxib. The patients, all male, were between 38 and 74 years. A total of 130 cycles of CT were administered. Toxicity associated with celecoxib was minimal. The RR was 56.5%. Median TTP and OS were 8.6 and 11.3 months, respectively. These data suggest that celecoxib may safely be combined with EP for treatment of ES-SCLC. This combination showed a promising activity and, despite the safety concerns regarding celecoxib, it would be interesting to further evaluate this regimen.

Biomarkers of spinal cord injury and ensuing bladder dysfunction

During the acute phase of SCI, the extension and residual neurological deficits that will persist after the waning of the spinal shock period are difficult to estimate on clinical grounds. Therefore, objective biomarkers able to estimate the extension of the lesion and the degree of neurological recovery are of great importance. Research has been focused on the detection of structural neuronal and glial proteins that leak from damaged cells, inflammatory proteins recruited to remove necrotic debris and more accurate neuroimaging methods that are able to discriminate the extension and functional consequences of the SCI. Urinary biomarkers are also being investigated to estimate functional changes that typically affect bladder function following SCI which can endanger patients life in the long run. Future studies are needed to precisely characterize the composition and function of the glial scar that appears in the area of SCI and repeals axonal growth, therefore preventing axonal rewiring.

Beta-3 adrenergic receptor is expressed in acetylcholine-containing nerve fibers of the human urinary bladder: An immunohistochemical study

To identify in the human bladder the structures which express the Beta‐3 adrenoceptor (β3AR).

Influence of functional genetic polymorphism (−590C/T) in non-small cell lung cancer (NSCLC) development: The paradoxal role of IL-4

Lung cancer is the leading cause of death by cancer in the world, originating about 17.5% of total deaths from cancer (1.18 million). Inflammation plays an important role in the pathogenesis of lung cancer. IL-4 is an anti-inflammatory cytokine, which reduces the production of proinflammatory cytokines by monocytes and with direct antiproliferative effects in some tumors. The polymorphism -590C/T SNP is a C to T transition in the -590 position of the promoter region of the IL-4 gene. The aim of this study was to evaluate the influence of this polymorphism in the susceptibility to NSCLC. DNA was extracted from peripheral blood of 1060 individuals (391 patients diagnosed with NSCLC and a control group of 669 individuals without cancer). The characterization of IL-4 -590C/T genotypes was performed by PCR-RFLP (BsmFI). The -590C/T polymorphism genotypes were classified as low (CC) and high expression (TT). The frequencies obtained for the CC and TT genotypes were 90.1% and 9.9%, respectively, in the control group and 92.9% and 7.1%, respectively, in the case group. The analysis of the TT and CC genotype frequencies in the two groups showed a statistically significant difference in its distribution, indicating a protection of 80% for the development of NSCLC, type epidermoid in individuals with the TT genotype when compared with individuals with CC genotype (P=0.024, OR=0.221: 95% CI=0.053-0.928). We present for the first time that increased expression of IL-4 associated with the TT genotype may contribute to immune surveillance during NSCLC development.

Impairment of sensory afferents by intrathecal administration of botulinum toxin A improves neurogenic detrusor overactivity in chronic spinal cord injured rats

Spinal cord injury (SCI) often leads to neurogenic detrusor overactivity (NDO) due to sprouting of sensory afferents on the lumbosacral spinal cord. NDO is characterized by high frequency of voiding contractions and increased intravesical pressure that may lead to urinary incontinence. The latter has been described as one of the consequences of SCI that mostly decreases quality of life. Bladder wall injections of botulinum toxin A (Onabot/A) are an effective option to manage NDO. The toxin strongly impairs parasympathetic and sensory fibres coursing the bladder wall. However the robust parasympathetic inhibition may inhibit voiding contractions and cause urinary retention in patients that retain voluntary voiding. Here, we hypothesised that by restricting the toxin activity to sensory fibres we can improve NDO without impairing voiding contractions. In the present work, we assessed the effect of Onabot/A on sensory neurons in chronic (4weeks) SCI rats by injecting the toxin intrathecally (IT), at lumbosacral spinal cord level. This route of administration was shown before to have an effect on bladder pain and contractility in an animal model of bladder inflammation. We found that IT Onabot/A led to a significant reduction in the frequency of expulsive contractions and a normalization of bladder basal pressure while maintaining voiding contractions of normal amplitude. Cleavage of SNAP-25 protein occurred mainly at the dorsal horn regions where most of the bladder afferents end. Cleaved SNAP-25 was not detected in motor or preganglionic parasympathetic neurons. A significant decrease in CGRP expression, a peptide exclusively present in sensory fibres in the spinal cord, occurred at the L5/L6 segments and associated dorsal root ganglia (DRG) after Onabot/A injection in SCI animals. Onabot/A strongly increased the expression of ATF3, a marker of neuronal stress, in L5/L6 DRG neurons. Taken together, our results suggest that IT Onabot/A has a predominant effect on bladder sensory fibres, and that such effect is enough to control NDO following chronic SCI. The mechanism of action of Onabot/A includes not only the cleavage of SNAP-25 in sensory terminals but also impairment of basic cellular machinery in the cell body of sensory neurons.