Blocking systemic nitric oxide production alters neuronal activation in brain structures involved in cardiovascular regulation during polymicrobial sepsis

In a previous study, we concluded that overproduction of nitric oxide (NO) by inducible nitric Oxide synthase (iNOS) in the late phase of sepsis prevents hypothalamic activation, blunts vasopressin secretion and contributes to hypotension, irreversible shock and death. The aim of this follow-up study was to evaluate if the same neuronal activation pattern happens in brain structures related to cardiovascular functions. Male Wistar rats received intraperitoneal injections of aminoguanidine, an iNOS inhibitor, or saline 30 min before cecal ligation and puncture (CLP) or sham surgeries. The animals were perfused 6 or 24 h after the surgeries and the brains were removed and processed for Fos immunocytochemistry We observed an increase (P < 0.001) in c-fos expression 6 h after CLP in the area postrema (AP), nucleus of he tractus solitarius (NTS), ventral lateral medulla (VLM), locus coeruleus (LC) and parabrachial nucleus (PB). At 24 h after CLP, however, c-fos expression was strongly decreased in all these nuclei (P < 0.05), except for the VLM. Aminoguanidine reduced c-fos expression in the AP and NTS at 6 h after CLR but showed an opposite effect at 24 h, with an increase in the AP, NTS, and also in the VLM. No such effect was observed in the LC and PB at 6 or 24 h. In all control animals, c-fos expression was minimal or absent. We conclude that in the early phase of sepsis iNOS-derived NO may be partially responsible for the activation of brain structures related to cardiovascular regulation. During the late phase, however, this activation is reduced or abolished. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Persistent Impairment of Testicular Histology and Sperm Motility in Adult Rats Treated with Cisplatin at Peri-Puberty

Cisplatin is one of the most widely used and effective chemotherapeutic agents for the treatment of several human malignancies. This study evaluated the effects of peri-pubertal cisplatin administration on several reproductive end-points and the reversibility of these effects in adulthood. Peri-pubertal Wistar male rats (45 days old) were divided into two groups: control (saline 0.9%) and cisplatin (1 mg/kg/day, 5 days/week, for 3 weeks, i.p.). The study was conducted in two steps and evaluations were performed at ages of 66 (post-pubertal age) and 140 (adult age) days on: (i) organ weights, serum gonadotropins and testosterone levels, sperm counts, motility and morphology, testicular histomorphometry, spermatogenesis kinetics, Sertoli cell number and in situ detection of apoptotic germ cells and (ii) sexual behaviour, fertility and intratesticular testosterone. At the end of cisplatin therapy, rats showed reductions in sperm production and reserves, sperm with progressive movement, tubular diameter, intratesticular testosterone and fertility potential, but increased numbers of TUNEL-positive seminiferous tubules, immotile sperm and pre-implantation losses compared with control. Moreover, cisplatin-treated post-pubertal rats displayed impaired testicular histopathology and sexual behaviour. Serum gonadotropins and testosterone levels, sperm morphology, spermatogenesis kinetics and Sertoli cell number were comparable between experimental groups at both ages. Alterations found in post-puberty were recovered at adulthood, except for sperm motility and damage to testicular histology. The persistence of these cisplatin effects, despite the unaltered fertility after natural mating in rats, may have implications for reproductive function of young boys undergoing cancer therapy, given the lower reproductive efficiency in human beings compared with rats.

Comprehensive gene expression profiling in lungs of mice infected with Mycobacterium tuberculosis following DNAhsp65 immunotherapy

Background The continued increase in tuberculosis (TB) rates and the appearance of extremely resistant Mycobacterium tuberculosis strains (XDR-TB) worldwide are some of the great problems of public health. In this context, DNA immunotherapy has been proposed as an effective alternative that could circumvent the limitations of conventional drugs. Nonetheless, the molecular events underlying these therapeutic effects are poorly understood. Methods We characterized the transcriptional signature of lungs from mice infected with M. tuberculosis and treated with heat shock protein 65 as a genetic vaccine (DNAhsp65) combining microarray and real-time polymerase chain reaction analysis. The gene expression data were correlated with the histopathological analysis of lungs. Results The differential modulation of a high number of genes allowed us to distinguish DNAhsp65-treated from nontreated animals (saline and vector-injected mice). Functional analysis of this group of genes suggests that DNAhsp65 therapy could not only boost the T helper (Th)1 immune response, but also could inhibit Th2 cytokines and regulate the intensity of inflammation through fine tuning of gene expression of various genes, including those of interleukin-17, lymphotoxin A, tumour necrosis factor-cl, interleukin-6, transforming growth factor-beta, inducible nitric oxide synthase and Foxp3. In addition, a large number of genes and expressed sequence tags previously unrelated to DNA-therapy were identified. All these findings were well correlated with the histopathological lesions presented in the lungs. Conclusions The effects of DNA therapy are reflected in gene expression modulation; therefore, the genes identified as differentially expressed could be considered as transcriptional biomarkers of DNAhsp65 immunotherapy against TB. The data have important implications for achieving a better understanding of gene-based therapies. Copyright (C) 2008 John Wiley & Sons, Ltd.

Increase of fat oxidation and weight loss in obese mice caused by chronic treatment with human growth hormone or a modified C-terminal fragment

OBJECTIVE: To observe the chronic effects of human growth hormone (hGH) and AOD9604 (a C-terminal fragment of hGH) on body weight, energy balance, and substrate oxidation rates in obese (ob/ob) and lean C57BL/6Jmice. In vitro assays were used to confirm whether the effects of AOD9604 are mediated through the hGH receptor, and if this peptide is capable of cell proliferation via the hGH receptor. METHOD: Obese and lean mice were treated with hGH, AOD or saline for 14 days using mini-osmotic pumps. Body weight, caloric intake, resting energy expenditure, fat oxidation, glucose oxidation, and plasma glucose, insulin and glycerol were measured before and after treatment. BaF-BO3 cells transfected with the hGH receptor were used to measure in Vitro I-125-hGH receptor binding and cell proliferation. RESULTS: Both hGH and AOD significantly reduced body weight gain in obese mice. This was associated with increased in vivo fat oxidation and increased plasma glycerol levels (an index of lipolysis). Unlike hGH, however, AOD9604 did not induce hyperglycaemia or reduce insulin secretion. AOD9604 does not compete for the hGH receptor and nor does it induce cell proliferation, unlike hGH. CONCLUSIONS: Both hGH and its C-terminal fragment reduce body weight gain, increase fat oxidation, and stimulate lipolysis in obese mice, yet AOD9604 does not interact with the hGH receptor. Thus, the concept of hGH behaving as a pro-hormone is further confirmed. This data shows that fragments of hGH can act in a manner novel to traditional hGH-stimulated pathways.

Maintaining clearance in peritoneal dialysis

Numerous studies have now established that there is a strong association between small solute clearance and improved outcomes in peritoneal dialysis (PD) patients. Preservation of both renal and peritoneal clearances is therefore of paramount importance, although very few trials have satisfactorily addressed this critical issue. Observational studies have suggested that the groups most at risk of loss of residual renal function are women, non-whites, diabetic patients, patients with congestive cardiac failure, patients who experience frequent episodes of peritonitis and, possibly, patients treated with automated PD (APD). There have been no controlled trials of renoprotective therapies in PD patients, but reasonable strategies for preventing renal functional decline include avoidance of nephrotoxins and infection, maintenance of adequate blood pressure, abstinence from smoking and possibly administration of angiotensin-converting enzyme inhibitors and/or calcium channel blockers. In contrast, peritoneal small solute removal can be maximized by augmenting fill volume, increasing exchange frequency and using either long-dwell continuous ambulatory PD (CAPD) or short-dwell (APD) therapies to suit individual patients' transport characteristics. Tidal PD may additionally increase solute clearance, although studies have reported conflicting findings. Preservation of membrane function may be achieved by minimizing episodes of peritonitis and avoiding hypertonic glucose exchanges. Newer peritoneal dialysates, such as icodextrin, amino acids, bicarbonate-buffered solutions and aldehyde-poor fluids, are more biocompatible in experimental models of PD, but their long-term clinical safety and efficacy have not yet been established by clinical trials. Moreover, no trials have demonstrated an independent effect of peritoneal clearance on patient outcomes. Further studies determining the relative value of renal and peritoneal clearances are therefore urgently required in order to optimize dialytic adequacy for PD patients.

Icodextrin as salvage therapy in peritoneal dialysis patients with refractory fluid overload

Background Icodextrin is a high molecular weight, starch-derived glucose polymer, which is capable of inducing sustained ultrafiltration over prolonged (12–16 hour) peritoneal dialysis (PD) dwells. The aim of this study was to evaluate the ability of icodextrin to alleviate refractory, symptomatic fluid overload and prolong technique survival in PD patients. Methods A prospective, open-label, pre-test/post-test study was conducted in 17 PD patients (8 females/9 males, mean age 56.8 ± 2.9 years) who were on the verge of being transferred to haemodialysis because of symptomatic fluid retention that was refractory to fluid restriction, loop diuretic therapy, hypertonic glucose exchanges and dwell time optimisation. One icodextrin exchange (2.5 L 7.5%, 12-hour dwell) was substituted for a long-dwell glucose exchange each day. Results Icodextrin significantly increased peritoneal ultrafiltration (885 ± 210 ml to 1454 ± 215 ml, p < 0.05) and reduced mean arterial pressure (106 ± 4 to 96 ± 4 mmHg, p < 0.05), but did not affect weight, plasma albumin concentration, haemoglobin levels or dialysate:plasma creatinine ratio. Diabetic patients (n = 12) also experienced improved glycaemic control (haemoglobin Alc decreased from 8.9 ± 0.7% to 7.9 ± 0.7%, p < 0.05). Overall PD technique survival was prolonged by a mean of 11.6 months (95% CI 6.0–17.3 months). On multivariate Cox proportional hazards analysis, extension of technique survival by icodextrin was only significantly predicted by baseline net daily peritoneal ultrafiltration (adjusted HR 2.52, 95% CI 1.13–5.62, p < 0.05). Conclusions Icodextrin significantly improved peritoneal ultrafiltration and extended technique survival in PD patients with symptomatic fluid overload, especially those who had substantially impaired peritoneal ultrafiltration.

Immune response to Bacteroides forsythus in a murine model

A murine skin abscess model was used to study the immune response to an acute infection with Bacteroides forsythus. BALB/c mice were given subcutaneous injections of either viable or heat-killed B. forsythus, while a third sham-immunized control group received phosphate-buffered saline. Weights and lesion sizes were measured. Blood was collected from the heart and specific antibodies to B. forsythus measured by an ELISA. Swabs taken from the lesions and also from pooled blood were cultured anaerobically for viable B. forsythus. Viable B. forsythus-induced lesions reached maximum size at day 7. B. forsythus cells were recovered from lesions up to day 4 although none were cultured from blood samples. Heat-killed bacteria induced much smaller lesions. Serum antibody levels increased during the 9-day study period, being significantly higher in mice injected with viable compared with heat-killed B. forsythus. Antibody levels in sham control mice were significantly lower than those seen in the other two groups. These results showed that a subcutaneous injection of viable cells of B. forsythus elicited a pronounced abscess formation and induce higher levels of specific antibodies compared with that produced by an injection of dead bacteria. This suggests that, as with other periodontopathic organisms, this mouse model can be used to study the immune response to B. forsythus.

Growth hormone receptor and IGF-1 receptor immunoreactivity during orthodontic tooth movement in the prednisolone-treated rat

Bone remodeling during tooth movement is regulated by local and systemic factors. Two regulators of bone metabolism are growth hormone (GH) and insulin-like growth factor-I (IGF-1). Their effects are mediated via binding to GH receptor (GHR) and IGF-I receptor (IGF-IR) in target tissues. Corticosteroids may affect the activity of these growth factors. This study examined the effect of prednisolone on GHR and IGF-IR expression in dental tissues following orthodontic tooth movement. The corti ticosteroid-treated group (N = 6) was administered prednisolone ( 1 mg/kg,) daily and the control group (N = 6) received equivalent volumes of saline. An orthodontic force (30 g) was applied to the maxillary first molar. Animals were sacrificed 12 days postappliance insertion. Sagittal sections of the first molar were stained for GHR and IGF-IR immunoreactivity. GHR and IGF-IR cell counts were elevated following appliance-treatment. Orthodontic tooth movement appeared to up-regulate GHR and IGF-IR immunoreactivity, but this up-regulation was reduced following prednisolone treatment. The suppression of GHR and IGF-I immunoreactivity in steroid-treated animals infers the mechanism whereby bone resorption and deposition, necessary for orthodontic tooth movement, may be inhibited by prednisolone. However, at 12 days postappliance insertion. no difference in orthodontic tooth movement was observed following low-dose prednisolone treatment.

Effects of high-dose etidronate treatment on microdamage accumulation and biomechanical properties in beagle bone before occurrence of spontaneous fractures

We recently demonstrated that suppressed bone remodeling allows microdamage to accumulate and causes reductions in some mechanical properties. However, in our previous study, I year treatment with high-dose etidronate (EHDP) did not increase microdamage accumulation in most skeletal sites of dogs in spite of complete remodeling suppression and the occurrence of spontaneous fractures of ribs and/or thoracic spinous processes. This study evaluates the effects of EHDP on microdamage accumulation and biomechanical properties before fractures occur. Thirty-six female beagles, 1-2 years old, were treated daily for 7 months with subcutaneous injections of saline vehicle (CNT) or EHDP at 0.5 (E-low) or 5 mg/kg per day (E-high). After killing, bone mineral measurement, histomorphometry, microdamage analysis, and biomechanical testing were performed. EHDP treatment suppressed intracortical and trabecular remodeling by 60%-75% at the lower dose, and by 100% at the higher dose. Osteoid accumulation caused by a mineralization deficit occurred only in the E-high group, and this led to a reduction of mineralized bone mass. Microdamage accumulation increased significantly by two- to fivefold in the rib, lumbar vertebra, ilium, and thoracic spinous process in E-low, and by twofold in the lumbar vertebra and ilium in E-high. However, no significant increase in damage accumulation was observed in ribs or thoracic spinous processes in E-high where fractures occur following 12 months of treatment. Mechanical properties of lumbar vertebrae and thoracic spinous processes were reduced significantly in both E-low and E-high. These findings suggest that suppression of bone remodeling by EHDP allows microdamage accumulation, but that osteoid accumulation reduces production of microdamage. (Bone 29:271-278; 2001) (C) 2001 by Elsevier Science Inc. All rights reserved.

Effects of suppressed bone turnover by bisphosphonates on microdamage accumulation and biomechanical properties in clinically relevant skeletal sites in beagles

We recently demonstrated that suppression of bone remodeling allows microdamage to accumulate, leading to reduced bone toughness in the rib cortex of dogs. This study evaluates the effects of reduced bone turnover produced by bisphosphonates on microdamage accumulation and biomechanical properties at clinically relevant skeletal sites in the same dogs. Thirty-six female beagles, 1-2 years old, were divided into three groups. The control group was treated daily for 12 months with saline vehicle (CNT), The remaining two groups were treated daily with risedronate at a dose of 0.5 mg/kg per day (RIS), or alendronate at 1.0 mg/kg per day (ALN) orally, The doses of these bisphosphonates were six times the clinical doses approved for treatment of osteoporosis in humans. After killing, the L-1 vertebra was scanned by dual-energy X-ray absorptiometry (DXA), and the L-2 vertebra and right ilium were assigned to histomorphometry, The L-3 vertebra, left ilium, Th-2 spinous process, and right femoral neck were used for microdamage analysis. The L-4 vertebra and Th-1 spinous process were mechanically tested to failure in compression and shear, respectively. One year treatment with risedronate or alendronate significantly suppressed trabecular remodeling in vertebrae (RIS 90%, ALN 95%) and ilium (RIS 76%, ALN 90%) without impairment of mineralization, and significantly increased microdamage accumulation in all skeletal sites measured. Trabecular bone volume and vertebral strength increased significantly following 12 month treatment. However, normalized toughness of the L-4 vertebra was reduced by 21% in both RIS (p = 0.06) and ALN (p = 0.05) groups. When the two bisphosphonate groups were pooled in a post hoc fashion for analysis, this reduction in toughness reached statistical significance (p = 0.02), This study demonstrates that suppression of trabecular bone turnover by high doses of bisphosphonates is associated with increased vertebral strength, even though there is significant microdamage accumulation and a reduction in the intrinsic energy absorption capacity of trabecular bone. (C) 2001 by Elsevier Science Inc. All rights reserved.

Growth hormone is permissive for skeletal adaptation to mechanical loading

The Lewis dwarf (DW) rat was used as a model to test the hypothesis that growth hormone (GH) is permissive for new bone formation induced by mechanical loading in vivo. Adult female Lewis DW rats aged 6.2 +/- 0.1 months (187 +/- 18 g) were allocated to four vehicle groups (DW), four GH treatment groups at 32.5 mug/100 g body mass (DWGH1), and four GH treatment groups at 65 mug/100 g (DWGH2). Saline vehicle or GH was injected intraperitoneally (ip) at 6:30 p.m. and 6:30 a.m. before mechanical loading of tibias at 7:30 a.m. A single period of 300 cycles of four-point bending was applied to right tibias at 2.0 Hz, and magnitudes of 24, 29, 38, or 48N were applied. Separate strain gauge analyses in 5 DW rats validated the selection of loading magnitudes. After loading, double-label histomorphometry was used to assess bone formation at the periosteal surface (Ps.S) and endocortical surface (Ec.S) of tibias. Comparing left (unloaded) tibias among groups, GH treatment had no effect on bone formation. Bone formation in tibias in DW rats was insensitive to mechanical loading. At the Ec.S, mechanically induced lamellar bone formation increased in the DWGH2 group loaded at 48N (p < 0.05), and no significant increases in bone formation were observed among other groups. The percentage of tibias expressing woven bone formation (Wo.B) at the Ps.S was significantly greater in the DWGH groups compared with controls (p < 0.05). We concluded that GH influences loading-related bone formation in a permissive manner and modulates the responsiveness of bone tissue to mechanical stimuli by changing thresholds for bone formation.

The effects of interleukin-10 depletion in vivo on the immune response to Porphyromonas gingivalis in a murine model

Background: The immune response to Porphyromonas gingivalis in the mouse abscess model is known to be dependent upon CD4 T-cell activation and the regulatory role of cytokines. The role of interleukin-10 (IL-10) in this mouse model was examined in vivo. Methods: One-week-old, female BALB/c mice were divided into 4 groups. Groups 1 and 2 were given intraperitoneal (ip) injections of phosphate buffered saline (PBS) weekly for 5 weeks. Group 3 was given an ip injection of rat immunoglobulin. Group 4 was injected with rat anti-IL-10 antibodies. At week 6, group 1 was sham-immunized with PBS, and groups 2, 3, and 4 were injected with P gingivalis lipopolysaccharide (Pg-LPS) weekly for 2 weeks. One week after the final immunization, delayed-type hypersensitivity (DTH) was assessed by footpad swelling to Pg-LPS. The level of serum antibodies to Pg-LPS and IFN-gamma (IFN-gamma) was determined by enzyme-linked immunosorbent assay. Dorsal abscess formation induced by the injection of viable P gingivalis was examined daily for 30 days. Results: The footpad swelling of the anti-IL-10-treated group (group 4) was significantly higher than that of groups 1 to 3. Similarly, the serum IFN-gamma level in group 4 was much higher than that of the other experimental groups. There was no significant difference in serum IgG antibodies to Pg-LPS in any of the experimental groups. However, the level of IgM antibodies in group 4 mice was significantly lower than that in groups 2 and 3. In addition, serum IgG1 was suppressed in group 4 mice, while IgG2a antibodies were raised. However, there was no difference observed between the levels of IgG2b and IgG3 antibodies in any group of mice. The lesions in sham-immunized mice (group 1) persisted for 30 days, and those in group 2 and 3 were undetected by day 18 and 20, respectively. In sharp contrast, lesions in group 4 had healed completely by day 13. Conclusions: This study has shown that IL-10 depletion in vivo in P gingivalis LPS-induced immune response in mice led to an elevated DTH response, an increase in serum IFN-gamma levels, and raised levels of IgG and IgG2a antibodies. Treatment with anti-IL-10 antibodies resulted in suppressed IgG I and IgM responses and a more rapid healing of abscesses than in non-IL-10-depleted mice. These results suggest that IL-10 depletion in Pg-LPS-induced immune response in mice may lead to a Th1-like immune response and provide strong protection against a subsequent challenge with live P gingivalis in an abscess model.

Early rise in blood pressure following administration of adrenocorticotropic hormone-[1-24] in humans

1. An elevation in blood pressure has been consistently observed 24 h after adrenocorticotropic hormone (ACTH) administration and is caused by increased ACTH-stimulated cortisol secretion, in association with increased cardiac output. The aim of the present study was to investigate the previously undefined time of onset of this increase in blood pressure in normal humans. 2. Ten normal healthy volunteers received 250 mug ACTH-[1-24], in 500 mL normal saline, infused at a constant rate over 8 h. Six subjects also received a placebo infusion (normal saline only). Blood pressure, heart rate and cortisol levels were determined hourly. Adrenocorticotropic hormone (ACTH-[1-24] plus native ACTH) was measured at 0, 1, 7 and 8 h. 3. Infusion of ACTH-[1-24] produced maximal secretion rates of cortisol, resulting in a mean peak plasma level of 985 +/- 46 nmol/L at 8 h. In response, blood pressure and heart rate rose significantly by 2 h and remained generally elevated for the duration of the infusion. 4. The early onset of haemodynamic responses is consistent with classical steroid receptor-mediated genomic mechanisms, but could be due non-genomic mechanisms. 5. The cardiovascular consequences of therapeutic use of ACTH are well recognized. This results of the present study suggest that even diagnostic administration of ACTH, delivered over a few hours, may raise blood pressure.

Effect of chronic clonidine treatment on transmitter release from sympathetic varicosities of the guinea-pig vas deferens

1 Previous studies have demonstrated that chronic pre-synaptic inhibition of transmitter release by morphine evokes a counter-adaptive response in the sympathetic nerve terminals that manifests itself as an increase in transmitter release during acute withdrawal. In the present study we examined the possibility that other pre-synaptically acting drugs such as clonidine also evoke a counter-adaptive response in the sympathetic nerve terminals. 2 In chronically saline treated (CST) preparations, clonidine (0.5 muM) completely abolished evoked transmitter release from sympathetic varicosities bathed in an extracellular calcium concentration ([Ca2+](o)) of 2 mM. The inhibitory effect of clonidine was reduced by increasing [Ca2+](o) from 2 to 4 mM and the stimulation frequency from 0.1 to 1 Hz. 3 The nerve terminal impulse (NTI) was not affected by concentrations of clonidine that completely abolished evoked transmitter release. 4 Sympathetic varicosities developed a tolerance to clonidine (0.5 muM) following 7-9 days of chronic exposure to clonidine. 5 Acute withdrawal of preparations following chronic clonidine treatment (CCT) resulted in a significant (P

Effect of chronic morphine treatment on alpha(2)-adrenoceptor mediated autoinhibition of transmitter release from sympathetic varicosities of the mouse vas deferens

1 The effect of chronic morphine treatment (CMT) on sympathetic innervation of the mouse vas deferens and on alpha (2)-adrenoceptor mediated autoinhibition has been examined using intracellular recording of excitatory junction potentials (EJPs) and histochemistry. 2 In chronically saline treated (CST) preparations. morphine (1 muM) and the alpha (2)-adrenoceptor agonist (clonidine, 1 muM) decreased the mean amplitude of EJPs evoked with 0.03 Hz stimulation by 81+/-8% (n=16) and 92+/-6% (n=7) respectively. In CMT preparations, morphine (1 muM) and clonidine (1 muM) decreased mean EJP amplitude by 68+/-8% (n = 7) and 79+/-8% (n = 7) respectively. 3 When stimulating the sympathetic axons at 0.03 Hz. the mean EJP amplitude recorded from smooth muscles acutely withdrawn from CMT was four times greater than for CST smooth muscles (40.7+/-3.8 mV, n = 7 compared with 9.9+/-0.3 mV, n = 7). 4 Part of the increase in mean EJP amplitude following CMT was produced by a 31% increase in the density of sympathetic axons and varicosities innervating the smooth muscle. 5 Results from the present study indicate that the effectiveness of alpha (2)-adrenocrptor mediated autoinhibition is only slightly reduced in CMT preparations. Most of the cross tolerance which develops between morphine, clonidine and alpha (2)-adrenoceptor mediated autoinhibition occurs as a consequence of increased efficacy of neuromuscular transmission which is produced by an increase in the probability of transmitter release and an increase in the density of sympathetic innervation.