Dendritic cells and interleukin 12 as adjuvants for tumor-specific vaccines.
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Adjuvants, Immunologic↗
Biomedical subjects
R Bianchi
Publications and source records attributed to R Bianchi.
Reduction of urinary 8-epi-prostaglandin F2 alpha during cyclo-oxygenase inhibition in rats but not in man.
1. 8-epi-prostaglandin (PG) F2 alpha, a major F2 isoprostane, is produced in vivo by free radical-dependent peroxidation of lipid-esterified arachidonic acid. Both cyclo-oxygenase isoforms (COX-1 and COX-2) may also form free 8-epi-PGF2 alpha as a minor product. It has been recently seen in human volunteers that the overall basal formation of 8-epi-PGF2 alpha in vivo is mostly COX-independent and urinary 8-epi-PGF2 alpha is therefore an accurate marker of 'basal' oxidative stress in vivo. 2. To test the validity of this marker in the rat, we evaluated in vivo the effect of COX inhibition on the formation of 8-epi-PGF2 alpha vs prostanoids. Two structurally unrelated COX inhibitors (naproxen: 30 mg kg-1 day-1; indomethacin: 4 mg kg-1 day-1) were given i.p. to rats kept in metabolic cages. In vivo formation of 8-epi-PGF2 alpha was assessed by measuring its urinary excretion. Prostanoid biosynthesis was assessed by measuring urinary excretion of major metabolites of thromboxane (TX) and prostacyclin (2,3-dinor-TXB1 and 2,3-dinor-6-keto-PGF1 alpha). All compounds were selectively measured by immunopurification/gas chromatography-mass spectrometry. 3. Naproxen reduced urinary excretion of 2,3-dinor-TXB1 and 2,3-dinor-6-keto-PGF1 alpha but, unexpectedly, also that of 8-epi-PGF2 alpha (82, 49 and 52% inhibition, respectively). Indomethacin had a similar effect (77, 69 and 55% inhibition). Esterified 8-epi-PGF2 alpha in liver and plasma remained unchanged after indomethacin. 4. These findings prompted us to re-assess the contribution of COX activity to the systemic production of 8-epi-PGF2 alpha in man. We gave naproxen (1 g day-1) to healthy subjects (four nonsmokers and four smokers). Urinary 8-epi-PGF2 alpha remained unchanged in the two groups (9.63 +/- 0.99 before vs 10.24 +/- 1.01 after and 20.14 +/- 3.00 vs 19.03 +/- 2.45 ng h-1 1.73 m-2), whereas there was a marked reduction of major urinary metabolites of thromboxane and prostacyclin (about 90% for both 11-dehydro-TXB2 and 2,3-dinor-TXB2; > 50% for 2,3-dinor-6-keto-PGF1 alpha). 5. To investigate whether rat COX-1 produces 8-epi-PGF2 alpha more efficiently than human COX-1, we measured the ex vivo formation of 8-epi-PGF2 alpha and TXB2 simultaneously in whole clotting blood. Serum levels of 8-epi-PGF2 alpha and TXB2 were similar in rats and man. 6. We conclude that a significant amount of COX-dependent 8-epi-PGF2 alpha is present in rat but not in human urine under normal conditions. This implies that urinary 8-epi-PGF2 alpha cannot be used as an index of near-basal oxidant stress in rats. On the other hand, our data further confirm the validity of this marker in man.
Adult↗
Effects of hypoxia and severity of diabetes on Na,K-ATPase activity and arachidonoyl-containing glycerophospholipid molecular species in nerve from streptozotocin diabetic rats.
The pathogenesis of experimental diabetic neuropathy is associated with the development of endoneurial hypoxia. Exposure of normal rats to hypoxic conditions has previously been shown to reduce nerve conduction velocity. To study the biochemical effects of hypoxia further, streptozotocin-induced diabetic and age-matched nondiabetic rats were maintained in air containing 10% oxygen for nine weeks. As compared to nondiabetic rats kept in room air, sciatic nerve Na,K-ATPase activity was decreased 38% in nondiabetic, hypoxic rats and tended to be lower in diabetic animals maintained in a normoxic environment. However, the enzyme activity was unchanged in diabetic, hypoxic rats, suggesting the existence of an undefined compensatory interaction between these two conditions. Arachidonoyl-containing molecular species (ACMS) of phosphatidylcholine and phosphatidylethanolamine were substantially depleted in nerves from diabetic rats. Hypoxia alone also caused a lesser depletion of some but not all of these ACMS. However, the two conditions together did not produce a further decrease, consistent with the concept that the same mechanism is responsible for loss of ACMS in hypoxia and diabetes. To examine the effects of severity of diabetes on these parameters, groups of rats were injected with either 50 mg/kg or 100 mg/kg streptozotocin. The latter group was maintained by administration of minimal insulin doses and the experiment was terminated after 3 weeks. Serum glucose in rats that received the high dose of drug averaged 12% higher than in the low dose group. As compared to nondiabetic rats, Na,K-ATPase activity was reduced 32-36%, but there was no difference in activity between the two diabetic groups. However, there was a greater loss of ACMS in the more severely hyperglycemic rats. In rats that received comparable streptozotocin doses, measurement of ACMS depletion after 3, 9 and 32 weeks of diabetes revealed the loss is progressive with time. Thus, glycerophospholipid ACMS is a sensitive index of the severity and duration of experimental diabetic neuropathy.
Animals↗
Constitutive expression of heat shock proteins 70 and 90 in rat cerebellum.
Exposure to heat shock and other stressful conditions activates in cells of all organisms a specific genetic program. This enhances the synthesis of proteins with a protective role against cellular damage, called heat shock proteins (hsps). Furthermore, in the mammalian nervous system, a considerable amount of hsps is also synthesized under normal conditions suggesting that they play an important role in the metabolism of unstressed cells. In this study we analysed the constitutive expression of proteins belonging to the hsp70 and hsp90 family in the rat cerebellum using immunocytochemistry with specific monoclonal antibodies. Our results showed that an intense immunostaining was evident, but was restricted in certain distinct cerebellar areas only, while no differences in the distribution of the two hsps were found. The strongest response was detected in the Purkinje neurons but deep cerebellar nuclei were also positive. In no case glial cells were found to be reactive for hsps despite their strong response for specific markers like glial fibrillary acid protein (astrocytes) and cyclic nucleotide phosphodiesterase (oligodendrocytes). These data indicate that both the hsp70 and hsp90 family have fundamental physiological functions in cerebellar neurons while they seem to play only a minor role in the metabolism of glial cells.
Animals↗
Tumor-associated trypsin inhibitor (TATI) and renal function.
Tumor-associated trypsin inhibitor (TATI) is a low molecular weight protein employed as tumor marker. To evaluate the role of the kidney in the clearance of TATI we studied the relationship of serum TATI with the glomerular filtration rate (GFR), and for comparisons the relationships of beta 2-microglobulin (beta(2m)) and creatinine with GFR. Urine excretion and renal extraction of TATI were also determined. The decrease in GFR was accompanied by an increase in blood levels of TATI, beta(2m) and creatinine. Serum TATI increased 12.4 times in patients with renal failure (GFR < 20 ml/min) with respect to subjects with normal renal function (P < 0.001, non-parametric Mann-Whitney test), while beta(2m) increased 7.3 times (P < 0.001) and creatinine 4.7 times (P < 0.001). In patients with GFR 60 to 40 ml/min, only the increase in TATI was statistically significant (p < 0.005). Renal excretion of TATI was low but it increased progressively in renal failure. Renal extraction ranged from 13% to 41%, for a mean 24.87. These results suggest that TATI is handled by the kidney and that it is a snesitrive marker of reduction in renal function.
Adolescent↗
Tyrosine phosphorylation of myelin protein PO.
Po (M(r) 30 kDa), the major protein component of peripheral nervous system (PNS) myelin, is known to be phosphorylated by protein kinase C on serine residues at multiple sites. This study was conducted to assess whether other amino acids might be phosphorylated in the protein. Segments of rat sciatic nerve were incubated with 32P in either the presence or absence of phorbol ester. Labeled Po was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to partial acid hydrolysis. Upon separation of the hydrolysis products by either thin-layer electrophoresis or thin-layer chromatography, a radioactive spot was detected which comigrated with authentic phosphotyrosine. In other experiments, nerves were incubated with the tyrosine phosphatase inhibitors vanadate or vanadyl hydroperoxide (pervanadate). When the nerve homogenate proteins were separated on gels and probed with a monoclonal antibody to phosphotyrosine on Western blots, a positive immune reaction was obtained for a protein species which migrated with the same mobility as PO on Coomassie Blue-stained gels. In the absence of 2-mercaptoethanol, this immunoreactive band displayed increased mobility on gels which is characteristic of the migration pattern of Po. The same immunostaining results were obtained using a purified peripheral myelin fraction prepared from nerve homogenates. Furthermore, the positions of immunoreactive bands produced by anti-Po and antiphosphotyrosine antibodies coincided on the same immunoblot of myelin proteins and purified Po. These data indicate that one or more tyrosyl residues in Po can be phosphorylated in intact sciatic nerve.
Animals↗
S-100 (alpha and beta) binding peptide (TRTK-12) blocks S-100/GFAP interaction: identification of a putative S-100 target epitope within the head domain of GFAP.
Alignment of previously characterized S-100 (alpha and beta)-binding peptides (J. Biol. Chem. 270, 14651-14658) has enabled the identification of a putative S-100 target epitope within the head domain of glial fibrillary acidic protein (GFAP). The capacity of a known peptide inhibitor of S-100 protein (TRTK-12), homologous to this region, to perturb the interaction of S-100 (alpha and beta) and GFAP (J. Biol. Chem 268, 12669-12674) was investigated. Fluorescence spectrophotometry and chemical cross-linking analyses determined TRTK-12 to disrupt S-100:GFAP interaction in a dose- and Ca(2+_dependent manner. TRTK-12 also inhibited S-100's ability to block GFAP assembly and to mediate disassembly of preformed glial filaments. Each of these events was strictly dependent upon the presence of calcium and inhibitory peptide, maximal inhibition occurring at a concentration of TRTK-12 equivalent to the molar amount of S-100 monomer present. Together with our recent report demonstrating TRTK-12 also blocks the interaction of S-100 protein with the actin capping protein, CapZ, these results suggest TRTK-12 functions as a pleiotropic inhibitor of S-100 function. Availability of a functional inhibitor of S-100 will assist the further characterization of S-100 protein function in vitro and in vivo. Moreover, this report provides additional evidence supportive of a role for S-100 as a multi-faceted regulator of cytoskeletal integrity.
2-Naphthylamine↗
IL-12 is both required and sufficient for initiating T cell reactivity to a class I-restricted tumor peptide (P815AB) following transfer of P815AB-pulsed dendritic cells.
Delayed-type hypersensitivity responses, mediated by CD8+ cells and detected by skin test assay, occur in sensitized mice in response to challenge with a class I-restricted synthetic peptide related to a poorly immunogenic tumor rejection Ag, P815AB, of murine mastocytoma cells. Efficient priming for this response, which requires functional CD4+ cells and production of IFN-gamma in the host, is achieved by transfer of dendritic cells (DC) pulsed in vitro with a physical mixture of P815AB and T helper peptides, such as a class II-restricted synthetic peptide of tetanus toxin. We now show that the adjuvant effect of the T helper peptide was associated with the appearance of early and late IL-12 transcripts in the spleens of DC recipient mice, correlated with a late IFN-gamma response, and was negated by serologic ablation of endogenous IL-12 at the time of cell transfer. rIL-12, administered in vivo to the DC recipient mice, could substitute for the T helper peptide in initiating skin test reactivity following transfer of DC pulsed with P815AB alone, leading to Ag-specific production of IFN-gamma by CD4+ and CD8+ T cells. In vitro and in vivo cell depletion experiments suggested the following: 1) the exogenous IL-12 required both CD4+ and CD8+ cells for activity; 2) the immune response initiated by IL-12 relied on later production of IL-12 by the host; and 3) the early adjuvanticity of the exogenous IL-12 involved improved recognition of class II-restricted epitopes of this otherwise poorly immunogenic tumor peptide.
Adjuvants, Immunologic↗
Preoperative evaluation of D-dimer and CA 125 levels in differentiating benign from malignant ovarian masses.
Plasma levels of D-dimer (DD) and CA 125 were measured preoperatively in 121 patients with ovarian masses submitted to laparotomy. DD and CA 125 levels were higher in the 56 patients with epithelial ovarian cancer than in the 65 patients with benign ovarian disease (P < 0.0001). The logistic regression procedure showed that both DD assay (cutoff 416 ng/ml) and CA 125 assay (cutoff 65 U/ml) were significant predictive variables for malignancy (P = 0.0001). The concordance between predicted probabilities and observed responses was 75.7% for DD, 72.1% for CA 125, and 90.8% for Dd and CA 125. It is worth noting that DD was increased ( > 416 ng/ml) in 73% of FIGO stage I patients, whereas CA 125 was above 65 U/ml in only 33.3%. Sensitivity, specificity, positive predictive value, and negative predictive value of the tests in differentiating benign from malignant ovarian masses were as follows: 76.8, 93.8, 91.5, and 82.4%, respectively, for CA 125; 94.6, 76.9, 77.9, and 94.3%, respectively, for the combination CA 125 or DD; and 73.2, 100.0, 100.0, and 81.3%, respectively, for the combination CA 125 and DD. The combination CA 125 and DD seems to be a useful diagnostic tool to differentiate benign from malignant ovarian masses. Elevated preoperative levels of both antigens are invariably associated with a postsurgical diagnosis of epithelial ovarian cancer.
Adult↗
Simultaneous dobutamine stress echocardiography and dobutamine scintigraphy (99mTc-MIBI-SPET) for assessment of coronary artery disease.
BACKGROUND: Simultaneous dobutamine stress echocardiography (DSE) and 99Tc-MIBI-SPET (DMS) for the evaluation of the presence and the extent of coronary artery disease (CAD) were assessed for a head to head comparison regarding the diagnostic acuracy of the two rests. METHODS AND RESULTS: Forty-five consecutive patients (33 males and 12 females: 53 +/- 6.8 yr.) underwent exercise electrocardiography and simultaneous dobutamine stress echocardiography and MIBI-SPET imaging. Coronary angiography was performed in all patients (significant coronary stenosis > 50%). On the basis of the results of exercise electrocardiogram the pre-test probability for coronary artery disease (Diamond's algorithm) was low (45.6 +/- 12.7%). The overall specificity, sensitivity and predictive accuracy of Echo-dobutamine stress test for diagnosis of the presence or absence of CAD were: specificity 82%, sensitivity 76%, diagnostic accuracy 80%, positive predictive value 90%, negative predictive value 40%. The overall specificity, sensitivity and predictive accuracy of MIBI-SPET-dobutamine test for diagnosis of the presence or absence of CAD were: specificity 86%, sensitivity 87%, diagnostic accuracy 84%, positive predictive value 97%, negative predictive value 54%. MIBI-SPET-dobutamine test showed a significantly higher sensitivity in comparison with ECHO-dobutamine test (P < 0.05). CONCLUSION: Both noninvasive methods for the detection of CAD showed a good diagnostic accuracy. Nevertheless the SPET model showed a higher sensitivity in comparison with DSE model, essentially in the presence of a lower extent of CAD and during submaximal test.
Cardiotonic Agents↗
A retroviral peptide encoded by mutated env p15E gene is recognized by specific CD8+ T lymphocytes on drug-treated murine mastocytoma P815.
Highly immunogenic ("xenogenized") tumour variants appear after treatment of murine mastocytoma P815 with the triazene derivative DTIC, a phenomenon associated with the appearance of structurally abnormal p15E env proteins in the variant cells. In the present study, we have isolated and sequenced several p15E cDNA gene fragments amplified by means of polymerase chain reaction (PCR) from parental (P815) and xenogenized (P815/DTIC) tumour cells. Compared to known p15E sequences in parental cells, one p15E sequence from xenogenized cells presented three distinct nucleotide changes, one of which was apparently unique to P815/DTIC DNA and cDNA upon single-nucleotide primer extension assay. One major histocompatibility complex (MHC) class I-binding peptide, corresponding to a putative mutation in the p15E sequence, was tested in parallel with the parental peptide for recognition by P815/DTIC-specific cytotoxic T cells in vitro. The results suggested that the amino acid substitution at the relevant position of the p15E protein may produce an antigenic T cell epitope. By skin test assay of mice primed with either the synthetic peptide or P815/DTIC cells, evidence was obtained that the mutated peptide is immunogenic in vivo, and that the neoepitope is expressed by P815/DTIC cells. In accordance with previous data in the L5178Y/DTIC tumour model system, these findings reinforce the notion that xenogenization of tumour cells may result in the expression of class I-binding mutated peptides of retroviral origin.
Amino Acid Sequence↗
Intramural Myomectomy Using a Combination of Hysteroscopy and Laparoscopy
Thanks to advances in hysteroscopic surgery and improvements in instrumentation, hysteroscopy can now be performed for intramural myomas. Twelve women with submucous or intramural myomas with a diameter ranging from 3.5 to 5.8 cm received preoperative preparation with 5 months of gonadotropin-releasing hormone analogs. The uterine cavity was distended with sorbitol-mannitol solution infused by an electronic pump. Hysteroscopic resection was performed with a 26F resectoscope and electrosurgical monopolar unit under laparoscopic control in all patients. Four women required a second procedure to complete the resection of myoma. No major complications occurred.
Journal Article↗
Risks of Virus Transmission During Diagnostic Hysteroscopy
The high frequency of asymptomatic carriers of viral infections represents a major risk for transmission. Viral agents can be transmitted through blood or biologic fluids during diagnostic hysteroscopy. Routine disinfection methods to clean hysteroscopes cannot be considered adequate to prevent transmission. Hepatitis C and B viruses require 1-hour sterilization in glutaric aldehyde or gas sterilization (ethylene oxide) to be decontaminated. Human immunodeficiency virus decontamination of instruments is easier and safer. Blood test screening of patients positive for hepatitis B and C should be performed in all candidates for endoscopic gynecologic procedures to avoid the possibility of virus transmission.
Journal Article↗
Normalization of peripheral thyroid hormone metabolism induced by successful chronic amiodarone treatment in patients with ventricular arrhythmias.
Amiodarone, an antiarrhythmic agent, is also known to have important effects on the peripheral metabolism of thyroid hormones; the relationship between these two effects of the drug, however, is not well established. We tested the hypothesis that the antiarrhythmic effect of amiodarone might be mediated by its effect on the metabolism of thyroid hormones. Peripheral thyroid hormone metabolism was investigated using a double-tracer ([125I]-T4 and [131I]-T3) procedure in 10 normal volunteers and 10 euthyroid patients with complex ventricular arrhythmias before and during 6 months' amiodarone treatment. The underlying cardiac disease was coronary artery disease in four cases, dilated cardiomyopathy in three and idiopathic arrhythmias in three. In all but one patient with complex ventricular arrhythmias amiodarone treatment resulted in a reduction of > or = 80% of premature ventricular contractions and complete suppression of episodes of ventricular pairs or ventricular tachycardia. In all cases successful treatment with amiodarone was accompanied by normalization of all kinetic parameters: T4 to T3 conversion ratio and T3/T4 molar ratio of production decreased to mean values of 24.7 +/- 17.5% and 0.35 +/- 0.22% respectively, whereas T4 production rate increased (mean value 75.9 +/- 30.0 nmol day-1 m-2). Our kinetic data indicate that long-term therapy with amiodarone, when effective in suppressing cardiac arrhythmias, also reduces peripheral T4 to T3 conversion, hence restoring the normal peripheral thyroid hormone metabolic pattern. In conclusion, our study confirms that the antiarrhythmic action of amiodarone may be (at least partially) mediated by its action on thyroid hormone metabolism, and may justify the hypothesis that an altered peripheral metabolism of thyroid hormones may play a role in the pathogenesis of complex ventricular arrhythmias.
Aged↗
Immunohistochemical characterization of thymic macrophages in normal and treated rats: a differential sensitivity to cyclosporine A.
In the present study, a set of two monoclonal antibodies (TRPM1, TRPM2) was used to investigate the macrophage populations in the rat thymus and their different sensitivities to cyclosporine-A (CsA). With double immunohistochemical staining we demonstrated that, in the normal rat thymus, there are 3 populations of macrophages (TRPM1+, TRPM1/2+, TRPM2+), present in different proportions throughout the thymus. In the outer cortex TRPM1+ and TRPM1/2+ were present, but the TRPM1/2+ cells were more numerous. No TRPM2+ cells were observed in this area. The cortex and medulla showed all 3 types of cells with a majority of TRPM1/2+ cells. In the corticomedullary zone (CMZ) TRPM1/2+ and TRPM2+ macrophages were present in about equal proportion while only a few TRPM1+ cells were observed. After CsA treatment (for 21 days) profound changes occurred in the thymus; we observed a complete disappearance of the thymic medulla and a reduction in the total number of macrophages. The TRPM1+ macrophages had been eliminated, a few TRPM1/2+ cells were found while many of the cells were TRPM2+. The presence of the macrophages in different thymic areas suggests that they are a very heterogeneous population. The possible significance of the macrophage heterogeneity and the relationship to CsA sensitivity is discussed.
Animals↗
Detection of Vibrio anguillarum by a sandwich enzyme-linked immunosorbent assay performed with monoclonal antibodies.
Monoclonal antibodies (Mabs) against V. anguillarum were produced and characterized by Western blotting analysis, competitive binding assays and cross-reactivity tests. Their ability to detect V. anguillarum in a liquid culture was tested in a sandwich enzyme-linked immunosorbent assay (ELISA) performed with different combinations of these Mabs used as capture or tracer antibodies. One combination was selected as the most suitable for diagnostic applications, showing the highest sensitivity and specificity.
Animals↗
Cytoarchitecture of the abducens nucleus of man: a Nissl and Golgi study.
The abducens nucleus is a pontine nucleus directly involved in oculomotion through its connections with the lateral rectus muscle of the eye. The aim of the present study was to investigate the cytoarchitectural organization of the abducens nucleus in man. The data obtained showed that the nerve cell bodies were small, medium and large in size and polygonal, oval, round or spindle shaped. The cytoplasm of all neurons appeared basophilic due to clearly evident scattered Nissl granules. On the basis of the characteristics of the dendritic arborization, multipolar and fusiform cells were identified. The multipolar neurons showed four to eight primary dendrites which gave off a wide secondary ramification. The fusiform neurons showed two dendrites emerging from the opposite poles of the elongated nerve cell body. The dendrites of all the neurons were largely confined within the boundaries of the nucleus. This finding would suggest that the neuronal relationships of the abducens nucleus supplied by the afferent fibers which pass through or end within it take place almost completely inside the nucleus. The wider dendritic arborization shown by the multipolar cells would indicate the latter as the principal target fields for the afferent inputs.
Abducens Nerve↗
Injection site effects on the pharmacokinetics and glucodynamics of insulin lispro and regular insulin.
OBJECTIVE: The pharmacokinetics and glucodynamics of a new insulin analog, insulin lispro, and regular human insulin were compared and contrasted after subcutaneous administrations in femoral, deltoid, and abdominal injection sites. RESEARCH DESIGN AND METHODS: Single 0.2 U/kg doses of insulin lispro and regular insulin were administered to 12 healthy subjects in a six-way randomized crossover fashion. Each dose was given after an overnight fast in one of three injection sites: abdominal, deltoid, or femoral. Study drugs were given during a manual euglycemic glucose clamp. Blood samples were collected over the 12-h clamp for measurement of insulin-reactive components, with pharmacokinetic and glucodynamic measurements derived from these serum insulin and clamp measurements. RESULTS: Glucodynamic comparisons between insulin lispro and regular insulin showed a greater maximum infusion rate (Rmax) at an earlier time (TRmax), regardless of injection site. The total glucose infused (Gtot) showed nearly identical values between sites for insulin lispro. Regular insulin showed greater Gtot values from deltoid and femoral injections. When comparisons were made between drugs, regular insulin produced significantly greater Gtot, primarily driven by the increased Gtot from deltoid and femoral injections. Greater maximum serum insulin concentrations (Cmax) were experienced with insulin lispro at earlier times (tmax), regardless of the injection site (P < 0.001). Abdominal administrations produced the greatest Cmax values at the earliest tmax for both regular insulin and insulin lispro. Deltoid and femoral injections had lower Cmax values for both compounds. Shifts also occurred with tmax, although these shifts were much greater with regular insulin than with insulin lispro. Equivalent area under the curve (AUC) values were found when compared between compounds. CONCLUSIONS: Slower absorption from deltoid and femoral administrations resulted in an increased duration of action for both regular insulin and insulin lispro when compared to abdominal administration. However, notable increases in the onset of action were only apparent with regular insulin. The consistency with insulin lispro response from abdominal and extremity injection sites allows more potential sites for subcutaneous injection with an assured rapid response.
Abdomen↗