Canine trachealis muscle shortening and cartilage mechanics.

Canine trachealis muscle will shorten by 70% of resting length when maximally stimulated in vitro. In contrast, trachealis muscle will shorten by only 30-40% when stimulated in vivo. To examine the possibility that an elastic load applied by the tracheal cartilage contributes to the in vivo limitation of shortening, single pairs of sonomicrometry crystals were inserted into the trachealis muscle at the level of the fifth cartilage ring in five dogs. The segment containing the crystals was then excised and mounted on a tension-testing apparatus. Points on the active length-tension curve and the passive length-tension relation of the cartilage only were determined. The preload applied to the muscle before contraction varied from 10 to 40 g (mean 21 +/- 4 g). The afterload applied by the cartilage during trachealis contraction ranged from 13 to 56 g (30 +/- 6 g). The calculated elastic afterloads were substantial and appeared to be sufficient to explain the degree of shortening observed in four of the seven rings; in the remaining three rings, the limitation of shortening was greater than would be expected from the elastic load provided by the cartilage. Additional sources of loading and/or additional mechanisms may contribute to limited in situ shortening. In summary, tracheal cartilage applies a preload and an elastic afterload to the trachealis that are substantial and contribute to the limitation of trachealis muscle shortening in vivo.

Mechanism of therapeutic effectiveness of cefixime against typhoid fever.

beta-Lactams have been considered ineffective against organisms growing inside mammalian cells because of their poor penetration into cells. However, cefixime has been shown to be clinically effective against typhoid fever. The probable mechanism of therapeutic effectiveness of cefixime against typhoid fever was investigated using Salmonella enterica serovar Typhimurium instead of S. enterica serovar Typhi both in a cellular and in a mouse infection model. Cefixime was able to inhibit the growth of serovar Typhimurium inhabiting monocyte-derived THP-1 cells. Elongation of serovar Typhimurium in THP-1 cells was observed microscopically. Apparent morphological changes of serovar Typhimurium in THP-1 cells were also observed by electron microscopy. The concentration of cefixime inside THP-1 cells was almost half (46 to 48%) of the concentration outside the cells when serovar Typhimurium coexisted in the solution. The length of time after oral dosing (8 mg/kg) that cefixime was present-calculated from levels in serum-at a concentration above the MIC at which 90% of the serovar Typhi organisms inside human cells were inhibited was presumed to be more than 12 h. Cefixime also showed excellent activity in the mouse systemic and oral infection models based on infections caused by serovar Typhimurium. It is concluded that a fair amount of cefixime can enter mammalian cells and inhibit the growth of bacteria inside cells when the bacteria are sensitive enough to cefixime, as are serovars Typhimurium and Typhi.

Effective cancer targeting using an anti-tumor tissue vascular endothelium-specific monoclonal antibody (TES-23).

Immunoconjugate targeting of solid tumors has not been routinely successful because the endo-thelial cells of blood vessels act as a physical barrier against the transport of macromolecules, such as antibodies. In the present study, we attempted to achieve tumor vascular targeting with an anti-tumor tissue endothelium-specific monoclonal antibody (TES-23). TES-23, an IgG1 monoclonal antibody raised against rat KMT-17 fibrosarcoma-derived endothelial cells, was covalently conjugated with neocarzinostatin (NCS) in a previous study. The TES-23-NCS conjugate induced tumor hemorrhagic necrosis, and showed marked anti-tumor effects against rat KMT-17 fibrosarcoma. This result prompted us to investigate whether this approach would be applicable to various other types of solid tumors. One hour after injection of (125)I-labeled TES-23 into BALB / c mice bearing Meth-A fibrosarcoma and Colon 26 adenocarcinoma, the tumor accumulation of TES-23 was greater than that of the control IgG. In the present study, we report the anti-tumor effects of this monoclonal antibody in mice bearing Meth-A fibrosarcoma. Mice treated with the immunoconjugate showed improved survival with no side effects. This result indicates that common antigens may be found in different kinds of tumor endothelial cells, and that TES-23 might recognize these antigens.

Purification and characterization of isoamyl acetate-hydrolyzing esterase encoded by the IAH1 gene of Saccharomyces cerevisiae from a recombinant Escherichia coli.

The IAH1 gene of Saccharomyces cerevisiae encodes an esterase that preferentially acts on isoamyl acetate; however, the enzyme has not yet been completely purified from the yeast S. cerevisiae. We constructed the IAH1 gene expression system in Escherichia coli, and purified the IAH1 gene product (Iah1p). The amount of Iah1p produced by recombinant E. coli was more than 40% of total cellular proteins. The molecular size of Iah1p was 28 kDa by SDS-polyacrylamide gel electrophoresis. Judging from the molecular weight estimation by gel filtration of purified Iah1p, the enzyme was thought to be a homodimer. The Km values for isoamyl acetate and isobutyl acetate were 40.3 mM and 15.3 mM, respectively. The enzyme activity was inhibited by Hg2+, p-chloromercuribenzoate, and diisopropylfluorophosphate.

Efficacy of FK463, a new lipopeptide antifungal agent, in mouse models of disseminated candidiasis and aspergillosis.

The efficacy of intravenous injection of FK463, a novel water-soluble lipopeptide, was evaluated in mouse models of disseminated candidiasis and aspergillosis and was compared with those of fluconazole (FLCZ) and amphotericin B (AMPH-B). In the candidiasis model, FK463 significantly prolonged the survival of intravenously infected mice at doses of 0.125 mg/kg of body weight or higher. In disseminated candidiasis caused by Candida species, including FLCZ-resistant Candida albicans, FK463 exhibited an efficacy 1.4 to 18 times inferior to that of AMPH-B, with 50% effective doses (ED(50)s) ranging from 0.21 to 1.00 mg/kg and 0.06 to 0.26 mg/kg, respectively, and was much more active than FLCZ. The protective effect of FK463 was not obviously influenced by the fungal inoculum size, the starting time of the treatment, or the immunosuppressed status of the host. The reduction in efficacy was less than that observed with FLCZ or AMPH-B. The efficacy of FK463 was also evaluated in the disseminated candidiasis target organ assay and was compared with those of FLCZ and AMPH-B. Efficacies were evaluated on the basis of a comparison between the mean log(10) CFU in kidneys in the groups treated with antifungal agents and that in control group. A single dose of FK463 at 0.5 mg/kg or higher significantly reduced the viable counts in kidneys compared with the numbers of yeast cells before treatment, and its efficacy was comparable to that of AMPH-B, while FLCZ at 4 mg/kg showed only a suppressive effect on the growth of C. albicans in the kidneys. In the disseminated aspergillosis model, FK463 given at doses of 0.5 mg/kg or higher significantly prolonged the survival of mice infected intravenously with Aspergillus fumigatus conidia. The efficacy of FK463 was about 2 times inferior to that of AMPH-B, with ED(50)s ranging from 0.25 to 0.50 mg/kg and 0.11 to 0.29 mg/kg, respectively. These results indicate that FK463 may be a potent parenterally administered therapeutic agent for disseminated candidiasis and aspergillosis.

Efficacy of FK463, a new lipopeptide antifungal agent, in mouse models of pulmonary aspergillosis.

The efficacy of FK463, a novel water-soluble lipopeptide, was evaluated in mouse models of pulmonary aspergillosis and was compared with that of amphotericin B (AMPH-B). In the pulmonary aspergillosis models induced by intranasal inoculation, FK463 exhibited good efficacy, with 50% effective doses in the range of 0. 26 to 0.51 mg/kg of body weight; these values were comparable to those of AMPH-B. In an Aspergillus target organ assay with immunosuppressed mice, under conditions of constant plasma levels of FK463, using a subcutaneously implanted osmotic pressure pump, a significant reduction in viable fungal cells was observed at plasma FK463 levels of 0.55 to 0.80 microgram/ml or higher. We conclude that FK463 is highly effective in the treatment of pulmonary aspergillosis in this animal model. These results indicate that FK463 may be a potent parenterally administered antifungal agent for pulmonary aspergillosis.

Specific binding of TES-23 antibody to tumour vascular endothelium in mice, rats and human cancer tissue: a novel drug carrier for cancer targeting therapy.

The tissue distribution of anti-tumour vascular endothelium monoclonal antibody (TES-23) produced by immunizing with plasma membrane vesicles from isolated rat tumour-derived endothelial cells (TECs) was assessed in various tumour-bearing animals. Radiolabelled TES-23 dramatically accumulated in KMT-17 fibrosarcoma, the source of isolated TECs after intravenous injection. In Meth-A fibrosarcoma, Colon-26 adenocarcinoma in BALB/c mice and HT-1080 human tumour tissue in nude mice, radioactivities of 125I-labelled TES-23 were also up to 50 times higher than those of control antibody with little distribution to normal tissues. The selective recognition of TES-23 to TECs was competitively blocked by preadministration of unlabelled TES-23 in vivo. Furthermore, immunostaining of human tissue sections showed specific binding of TES-23 on endothelium in oesophagus cancers. These results indicate that tumour vascular endothelial cells express common antigen in different tumour types of various animal species. In order to clarify the efficacy of TES-23 as a drug carrier, an immunoconjugate, composed of TES-23 and neocarzinostatin, was tested for its anti-tumour effect in rats bearing KMT-17 fibrosarcomas. The immunoconjugate (TES-23-NCS) caused marked regression of the tumour, accompanied by haemorrhagic necrosis. Thus, from a clinical view, TES-23 would be a novel drug carrier because of its high specificity to tumour vascular endothelium and its application to many types of cancer.

In vivo antibacterial activity of FK041, a new orally active cephalosporin.

The therapeutic activities of orally administered FK041 were evaluated in mouse models of systemic and local infections with a variety of bacteria and were compared with those of cefdinir (CFDN) and cefditoren pivoxil (CDTR-PI). FK041 exhibited potent therapeutic activity against lethal systemic infections induced by intraperitoneally inoculated Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae with 50% effective doses (ED50) in the range of 0.20 to 0.36 mg/kg and was more active than CFDN and CDTR-PI. This result correlated well with its in vitro activity. The therapeutic effects of FK041 and reference drugs on murine local infections were evaluated in an in vivo pharmacokinetic model simulating human plasma concentrations for oral administration of 50 mg, 100 mg, and 200 mg. Against murine subcutaneous abscess induced by S. aureus, FK041 was as effective as CFDN and significantly more effective than CDTR-PI in reducing the number of recoverable viable bacteria in the skin at the infection sites. The efficacy of FK041 against murine pneumonia with H. influenzae was comparable to that of CDTR-PI and was superior to that of CFDN in reducing viable bacteria activity in the lungs. These results strongly suggest that FK041 has potential for clinical use against various bacterial infections.

Suppression of solid tumor growth by a monoclonal antibody against tumor vasculature in rats: involvement of intravascular thrombosis and fibrinogenesis.

We have reported that immunization of rat tumor-derived endothelial cells (TEC) isolated from KMT-17 solid tumors results in the generation of several monoclonal antibodies (MAbs). TES-23, one of these MAbs, recognizes a naturally occurring 80-kDa antigen expressed on endothelial cells of tumor blood vessels. To determine whether such MAbs can suppress solid tumor growth in vivo by impairment of endothelial cells in tumors following direct binding, we tested the biodistribution of (125)I-labeled TES-23 in rats bearing KMT-17 solid tumors. We also examined the effect of treatment using unconjugated TES-23 on tumor growth and histo-pathological changes in tumor tissues. Biodistribution studies showed localization of TES-23 into tumor tissues 60 min after intravenous injection. TES-23 suppressed significantly the growth of KMT-17 solid tumors following administration for 5 days. Histo-pathological examination showed that TES-23 caused degeneration, apoptosis and/or necrosis and denudation of endothelial cells in viable tumor areas following local aggregation and adhesion of lymphocytes, with subsequent intravascular thrombus formation by platelets and fibrin. Our results indicate that TES-23, which recognizes TEC, can target endothelial cells of solid tumor vasculature directly, resulting in growth suppression in vivo by reduction of blood flow due to intravascular thrombosis. Our results also suggest that targeting tumor vasculature is a potentially attractive approach for the treatment of solid tumors.

Tumor vascular targeting using a tumor-tissue endothelium-specific monoclonal antibody as an effective strategy for cancer chemotherapy.

In this study, we attempted to develop tumor vascular targeting with a tumor tissue endothelium-specific monoclonal antibody. TES-23, which strongly and selectively recognizes tumor tissue endothelial cells, was chemically conjugated with Neocarzinostatin (NCS), and the anti-tumor effect was examined. The immunoconjugate, TES-23-NCS, showed, through the use of tumor hemorrhagic necrosis, a marked anti-tumor effect on KMT-17 tumors in rats at a dosage of 17 micrograms/kg (NCS equivalent) without any side effects, probably due to specific tumor vascular injury. By contrast, TES-23 alone (107 micrograms/kg), NCS alone (17 micrograms/kg), and Mopc-NCS (Mopc, 107 micrograms/kg; NCS, 17 micrograms/kg), the immunoconjugate of control antibody, did not have any anti-tumor activities. By tissue distribution analysis, TES-23 and TES-23-NCS showed high accumulation in KMT-17 tumors 1 h after intravenous administration. Moreover TES-23 also accumulated in Sarcoma-180 tumors in mice 1 h after intravenous administration. These results suggest that TES-23 may be a candidate for a potential tumor vascular targeting agent that is applicable to a wide variety of tumor types.

Balance of activities of alcohol acetyltransferase and esterase in Saccharomyces cerevisiae is important for production of isoamyl acetate.

Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash.

Identification of tumor vascular antigens by monoclonal antibodies prepared from rat-tumor-derived endothelial cells.

We have reported the isolation and specific in vitro properties of tumor-derived endothelial cells (TEC) from rat KMT-17 fibrosarcomas transplanted into rats. To develop antibody-based tumor vascular targeting therapy for solid tumors, we have generated monoclonal antibodies (MAbs) using passive immunization of outside-out membrane vesicles of rat epididymal-fat-pad-derived capillary endothelial cells (FCEC) followed by active immunization of those of rat TEC. The MAbs produced were screened against TEC and FCEC. Of all cultured hybridomas, 75 (3.3%) of the secreted MAbs preferentially recognized TEC. We selected a total of 7 MAbs which detected antigens highly abundant in TEC, although 5 of the 7 MAbs were weakly positive for FCEC in cell-ELISA and FACS analyses. The antigens recognized by these MAbs, with the exception of MAb TES-7, were present on endothelial cells of tumor blood vessels in KMT-17 fibrosarcoma tissues, as shown by immunohistochemical analysis. Antigens of 40- and 80-kDa were recognized by MAbs TES-1, 7, 17, 21 and 26 and by MAbs TES-23 and 27 respectively. Although the function of these antigens, which are preferentially expressed on rat tumor-derived endothelial cells, is still unknown, we believe that future studies of such antigens will help elucidate the role of endothelial cells in tumor vasculature. Our results indicate that MAbs may provide a novel tool for the development of antibody-based therapy targeting tumor vasculature.

Antibody-based therapy targeting tumor vascular endothelial cells suppresses solid tumor growth in rats.

We have developed a new approach to antibody-based therapy of solid tumors by targeting tumor vascular endothelial cells (EC) which are essential for the growth of solid tumors. We investigated the effect of an antibody against tumor-derived endothelial cells (TEC) on the growth of solid tumors in rats. Intravenous administration of TES-23, a monoclonal antibody generated by TEC isolated from rat KMT-17 solid tumors, at 1 mg/rat/day for 5 days resulted in significant suppression of KMT-17 tumor growth. Histopathological analysis of tumors administered with TES-23 showed that adhesion of lymphocytes to EC followed by denudation of EC in the viable tumor area. In contrast, little obvious toxicity was observed in most of the rat organs examined. These findings suggest that the concept of an antibody-based therapy with targeting tumor vascular EC would be promising in treatment of solid tumors.

Effect of tumour cell-conditioned medium on endothelial macromolecular permeability and its correlation with collagen.

Conditioned medium prepared from mouse melanoma B16 cells (B16-CM) increases the macromolecular permeability of bovine aortic, venous and human umbilical vein endothelial monolayer. Collagen, which is synthesised by endothelial cells, has an important function in regulating the permeability of endothelial monolayer. Briefly, low collagen content leads to hyperpermeable structure of the endothelial monolayer. In the present studies, we examined the relationship between the increase of endothelial permeability and content of synthesised collagen of endothelial cells cultured with B16-CM. The B16-CM reduced endothelial collagen content but did not digest collagen directly. Matrix metalloproteinase inhibitor, 1,10-phenanthroline, inhibited the increase in permeability due to addition of B16-CM. These data suggest that B16-CM acts on endothelial cells, stimulating the digestion of endothelial collagen, and that the reduced content of collagen leads to the hyperpermeability of the endothelial monolayer.

Isolation and properties of tumor-derived endothelial cells from rat KMT-17 fibrosarcoma.

Rat KMT-17 fibrosarcoma-derived endothelial cells were isolated by Percoll gradient centrifugation with an attaching-speed separation technique, and their properties in culture were examined. The primary cultured tumor-derived endothelial cells (TEC) showed angiotensin-converting enzyme activity, positivity for Factor VIII-related antigen staining, and typical capillary-like formation on Matrigel. The primary cultured TEC monolayer showed greater permeability than normal tissue-derived endothelial cell (aorta, vena cava and epididymal fat capillary) monolayers on FITC-dextran diffusion (molecular weight 70,000). Leukocyte adhesion to TEC was reduced compared to that to fat-derived capillary endothelial cells. These characteristics resembled those of tumor vascular endothelium, and were observed both in the primary and first-passage cell cultures, but not in the fourth-passage cell cultures. Our findings indicate that primary or subcultured TEC are applicable for studies of the physiological characteristics of tumor endothelial cells.

Regional diaphragm shortening measured by sonomicrometry.

Diaphragmatic shortening measured by sonomicrometry has been compared in the two major anatomic segments, costal and crural. Data obtained by videofluoroscopy found a variation in subsegmental shortening within segments (Sprung et al. J. Appl. Physiol. 67: 655-662, 1989). No reproducible pattern of subsegmental shortening has emerged, and the mechanisms leading to this subsegmental variation in shortening are unknown. Therefore, we compared subsegmental shortening in both segments of the diaphragm in seven supine pentobarbital-anesthetized dogs. Seven pairs of sonomicrometer transducers were implanted in the two segments, and subsegmental shortening during spontaneous breathing was measured. To determine potential mechanisms contributing to the variation in shortening, measurements were made during stimulated breathing, after epiphrenic stimulation, and during occluded breaths. We found electrical stimulation at physiological frequencies of 10 and 20 Hz reduced the variation in subsegmental shortening, whereas stimulated breathing did not. Occluded breaths showed a consistent decrease in the amount of shortening, particularly in the dome of the costal diaphragm, compared with shortening in the area of apposition. Comparison of shortening between segments revealed greater crural than costal shortening. We conclude that subsegmental variation in activation can contribute to variation in subsegmental shortening and that the afterload can effect shortening during occluded breaths.

Tumor necrosis factor alpha decreases in vivo diaphragm contractility in dogs.

In this study, we hypothesized that tumor necrosis factor alpha (TNF alpha) is an important mediator of sepsis-related impairment in diaphragm contractility (1-2). In 12 anesthetized, ventilated dogs, bipolar stimulating electrodes were placed on the phrenic nerves and diaphragm electromyographic activity (EMG) and shortening were recorded with needle electrodes and piezoelectric crystals, respectively. Transdiaphragmatic pressure (Pdi) was also recorded using esophageal (Pes) and abdominal balloon catheters (Pdi = Pab-Pes). Dogs were randomized to receive saline injection (n = 6), or TNF alpha 60 micrograms/kg (n = 6). All parameters were recorded hourly for 6 h. Mean arterial blood pressure decreased 1 h after infusion in TNF alpha animals (p < 0.05) with no significant change thereafter. Cardiac output increased early after TNF alpha infusion (p < 0.05) and remained at greater than baseline values at study termination. Diaphragm pressure generation and costal shortening decreased progressively from 3 to 6 h post TNF alpha infusion (p < 0.05) with no significant change in control animals. Compound diaphragm action potential in response to supramaximal phrenic stimulation decreased in TNF alpha animals (p < 0.01) with no significant change in control animals 3 and 6 h postinfusion. We conclude that TNF alpha infusion was associated with significant declines in isotonic and quasi-isometric diaphragm contraction and that this could be explained, at least in part, by impaired neuromuscular transmission.

Decrease in left ventricular contractility after tumor necrosis factor-alpha infusion in dogs.

Whether systolic contractility or diastolic compliance changes soon after tumor necrosis factor-alpha (TNF-alpha) exposure is not known. Accordingly, we measured hemodynamics, left ventricular contractility using the slope of the end-systolic pressure-volume relationship, and diastolic pressure-volume relationships in six control dogs and in six dogs receiving 60 micrograms.kg-1.h-1 i.v. of TNF-alpha. Mean aortic pressure decreased by 22% 1 h after TNF-alpha infusion and remained decreased (P < 0.05). Cardiac output increased by 19% 1 h after TNF-alpha infusion and remained significantly greater than control values (P < 0.05). Left ventricular contractility decreased by 23% (P < 0.05) 1 h after TNF-alpha infusion and decreased by 52% (P < 0.01) 5 h after TNF-alpha infusion. The diastolic pressure-volume relationship did not change in the TNF-alpha group or the control group. Ejection fraction did not change after TNF-alpha infusion despite the decrease in contractility because afterload decreased. We conclude that TNF-alpha is important in causing the hypotensive, hyperdynamic circulation of sepsis. The new finding that left ventricular contractility is decreased shortly after TNF-alpha infusion suggests that TNF-alpha, or another mediator released very soon after TNF-alpha, is an important myocardial depressant factor.

Simulation of human plasma levels of beta-lactams in mice by multiple dosing and the relationship between the therapeutic efficacy and pharmacodynamic parameters.

A mathematical multiple dosing model was designed so that human plasma concentration-versus-time curves of beta-lactams are reproduced in mouse plasma. The pharmacokinetic parameters of FK037, a new injective cephalosporin, in volunteers and in the mice model were 6,966 and 6,894 ml, respectively, for Vc, 2.592 and 2.698/h for alpha, 0.2875 and 0.3027/h for beta, and 0.9079 and 1.0506 for K21. Therefore, real pharmacokinetics of humans were reproduced in mice by this method. The 8-hour therapeutic efficacy (the decrease of the viable counts in the lung) against pneumonia with Staphylococcus aureus and Pseudomonas aeruginosa in mice was well correlated with the time above MIC value, but not with AUC, Cmax or AUC above MIC. These results indicate that this model was valuable to evaluate the beta-lactam antibiotics for predicting their clinical efficacy and that the time above MIC is an important factor in selecting beta-lactam agents and determining dosage in pulmonary infection.

Delayed poststimulus decrease of phrenic motoneuron output produced by phrenic nerve afferent stimulation.

The immediate effects of phrenic afferent nerve activation on ventilation have been shown to be both excitatory and inhibitory. Long-lasting inhibitory effects on respiratory motoneuron output have been reported after stimulation of afferent nerves from limb muscles. However, whether respiratory muscle afferent nerves can produce this effect is unknown. We therefore hypothesized that activation of phrenic afferent nerves may produce a prolonged decrease of respiratory motoneuron output. Six alpha-chloralose-anesthetized dogs were studied after vagotomy and bilateral carotid sinus nerve section. The dogs were paralyzed, and end-tidal CO2 was controlled by mechanical ventilation. The proximal end of the cut thoracic phrenic nerve was electrically stimulated for 1 min at intensities that produced activation of thin-fiber afferents. The contralateral efferent phrenic integrated electroneurogram (ENG) was recorded. During stimulation, phrenic ENG activity increased. ENG activity was recorded during recovery and reached a peak decrease compared with control of 19 +/- 11% (SD) 9.0 +/- 6 min after stimulation and returned to control after 30 min. A qualitatively similar response was seen after stimulation of the gastrocnemius nerve. We conclude that activation of thin-fiber afferents in the phrenic nerve can produce a delayed and prolonged decrease of respiratory motoneuron output similar to that of limb muscle afferent nerves.