Predicting adjacent infections in pediatric septic arthritis: Do predictive criteria extrapolate across geographic regions?: Predicting Periarticular Infection in the Southeast.

PURPOSE: This study aims to assess previously determined predictive criteria for presence of adjacent infection in septic arthritis within a Southeastern United States (US) pediatric population. RESULTS: The sensitivity, specificity, positive predictive value, and negative predictive value of the Rosenfeld criteria were: 91.7%, 22.7%, 39.3%, and 83.3%, respectively. The patients with periarticular infection were more likely to have positive blood cultures than those with isolated septic arthritis. There was no difference in likelihood of secondary surgical intervention. CONCLUSIONS: Previously defined criteria to predict adjacent infection in pediatric septic arthritis did not demonstrate external validity in a Southeastern US pediatric population.

Benzoyl Peroxide is Cost-Effective for Preventing Infection by Cutibacterium Acnes in Arthroscopic Rotator Cuff Repair.

PURPOSE: To evaluate the cost-effectiveness of benzoyl peroxide (BPO) in decreasing postoperative infections through a mathematical model in the setting of arthroscopic rotator cuff repair (RCR). METHODS: A break-even equation compared the costs associated with perioperative BPO use and postoperative infection following an arthroscopic RCR. The postoperative infection rate used for calculations was 0.28%, a value established in current literature. The break-even analysis produced a new infection rate, which defined how much BPO is needed to reduce the known infection rate in order for its prophylactic use to be cost-effective. The institution's business office assessed the minimum itemized costs associated with the standard-of-care treatment of postoperative RCR infection. Sensitivity analysis was conducted to demonstrate how variability in the costs of BPO, in infection rates and in the cost of infection treatment affected the absolute risk reduction (ARR) and number needed to treat (NNT). RESULTS: Financial review yielded a minimum institutional cost of treating a postoperative infection following arthroscopic RCR of $24,991.31. Using the break-even formula to calculate the ARR at which the overhead costs of BPO and the treatment of infection were equal, BPO was economically viable if it decreased infection rate by 0.000734% (NNT = 1,361.92). This value was low because of the order of magnitude of difference between the costs of infection prevention when compared to the costs of treating postoperative infections. CONCLUSIONS: This break-even analysis model suggests that the use of preoperative BPO in the setting of arthroscopic RCR is cost-effective for prevention of infection with Cutibacterium acnes, given the high cost of treating the infection versus the low cost of the solution. CLINICAL RELEVANCE: The economic feasibility of preoperative use of BPO in the setting of arthroscopic RCR could alter the standard of care.

Long-term effects of maternal undernutrition on offspring carotid artery remodeling: role of miR-29c.

The purpose of this study was to examine the hypothesis that excess maternal glucocorticoids in response to maternal undernutrition programs the expression of extracellular matrix (ECM) components potentially by miR-29c. We measured the expression of mRNA (qRT-PCR) and protein (Western blot) for collagen 3A1, collagen 4A5 and matrix metalloproteinase 2 (MMP2) in offspring carotid arteries from three groups of dams: 50% food-restricted in latter half of gestation [maternal undernutrition (MUN)], MUN dams who received metyrapone (MET) (500 mg/ml ) in drinking water from day 10 of gestation to term, and control dams fed an ad libitum diet. The expression of miR-29c was significantly decreased at 3 weeks, 3 months and 9 months in MUN carotid arteries, and these decreases in expression were partially blocked by treatment of dams with MET. The expression pattern of ECM genes that are targets of miR-29c correlated with miR-29c expression. Expression of mRNA was increased for elastin (ELN) and MMP2 mRNA in 3-week MUN carotids; in 9-month carotids there were also significant increases in expression of Col3A1 and Col4A5. These changes in mRNA expression of ECM genes at 3 weeks and 9 months were blocked by MET treatment. Similarly, the expression of ELN and MMP2 proteins at 3 weeks were increased in MUN carotids, and by 9 months there were also increases in expression of Col3A1 and Col4A5, which were blocked by MET in MUN carotids. Overall, the results demonstrate a close correlation between expression of miR-29c and the ECM proteins that are its targets thus supporting our central hypothesis.

Carotid artery reconstruction for infected carotid patches.

OBJECTIVES: Infected carotid prosthetic patches (ICPP) are a rare but catastrophic complication of carotid endarterectomy (CEA). Prevention and appropriate surgical management is essential. We report our experience of carotid artery reconstruction for ICPP. DESIGN: Single-center retrospective study. METHODS: 10-year review of the surgical treatment of ICPP. RESULTS: Twelve patients presented with patch infection following CEA. Three patients presented acutely with an expanding hematoma, eight with chronic complications (abscess/discharging sinus n = 5, carotid pseudoaneurysm n = 3). Mean age was 75 years. Replacement conduits included superficial femoral artery (n = 6), cadaveric homograft (n = 3), long saphenous vein (n = 2) and one patient had primary closure. Five patients had muscle flaps fashioned for carotid artery protection. Operative complications included hypoglossal nerve injury (1 patient), superficial skin infection (2 patients) and one patient was returned to the operating room for a neck haematoma. Five surgical specimens were culture positive for: Staphylococcus aureus (n = 3), Corynebacterium propionibacterium (n = 1) and Streptococcus anginous (n = 1). There were no 30-day mortalities. Mean hospital stay was 6 days. Median follow-up was 16 months (range 3-108 months). CONCLUSION: Carotid artery reconstruction in a contaminated wound represents a significant surgical challenge. Unlike previous reports that used venous conduits, this is the first series where cadaveric or autologous arterial conduits were preferred. Arterial conduits achieved durable short term follow-up.

Effect of maternal undernutrition on vascular expression of micro and messenger RNA in newborn and aging offspring.

The aim of this study was to test the hypothesis that maternal undernutrition (MUN) alters offspring vascular expression of micro-RNAs (miRNAs), which, in turn, could regulate the expression of a host of genes involved with angiogenesis and extracellular matrix remodeling. The expression of miRNA and mRNA in the same aortic specimens in 1-day-old (P1) and 12-mo-old offspring aortas of dams, which had 50% food restriction from gestation day 10 to term, was determined by specific rat miRNA and DNA arrays. MUN significantly downregulated the expression of miRNAs 29c, 183, and 422b in the P1 group and 200a, 129, 215, and 200b in the 12-mo group, and upregulated the expression of miRNA 189 in the P1 group and 337 in the 12-mo group. The predicted target genes of the miRNAs altered in the two age groups fell into the categories of: 1) structural genes, such as collagen, elastin, and enzymes involved in ECM remodeling; and 2) angiogenic factors. MUN primarily altered the expression of mRNAs in the functional category of cell cycle/mitosis in the P1 group and anatomic structure and apoptosis in the 12-mo age group. Several of the predicted target genes of miRNAs altered in response to MUN were identified by the DNA array including integrin-beta(1) in the P1 aortas and stearoyl-CoA desaturase-1 in the 12-mo age groups. These results are consistent with the hypothesis that MUN modulation of offspring gene expression may be mediated in part by a miRNA mechanism.

Autologous peripheral blood CD133+ cell implantation for limb salvage in patients with critical limb ischemia.

We report the safety and feasibility of autologous CD133+ cell implantation into the lower extremity muscles of patients with critical limb ischemia, whose only other option was limb amputation. Nine patients participated in the study: seven patients suffering from arteriosclerosis obliterans, one with thromboangiitis obliterans (Buerger's disease) and one with thromboembolic disorder. Autologous PBSC were collected after the administration of G-CSF (10 mcg/kg/day). CD133+ cells were selected using the CLINIMACS cell separation device and were injected i.m. without earlier cryopreservation using a 22-gauge needle into multiple sites 3 cm apart in the gastrocnemius/soleus muscle, or depending on clinical circumstances, in the foot or quadriceps muscle, or both, of the involved leg. There were no complications from either leukapheresis or injection. Stem cell injection prevented leg amputation in seven of the nine patients. In this small cohort of patients with end-stage critical limb ischemia, quality of life (Short Form-36) physical component score improved significantly at 3 (P=0.02) and 6 (P=0.01) months, but not at 1 year (P=0.08). There was a trend towards the improvement in pain-free treadmill walking time (P=0.13) and exercise capacity (P=0.16) at 1 year. Lower extremity limb salvage was achieved for seven of the nine treated patients.

The soluble forms of CD28, CD86 and CTLA-4 constitute possible immunological markers in patients with abdominal aortic aneurysm.

OBJECTIVE: The T cell co-stimulatory factors CD28 and CTLA-4 and their ligands CD80 and CD86 occur as receptors on T cells and antigen-presenting cells and also in soluble forms in the circulation. We determined the levels of soluble co-stimulatory molecules in patients with abdominal aortic aneurysm (AAA) and normal individuals. We further correlated these soluble co-stimulatory molecules to other clinical parameters of importance such as age of the patient, presence of hypertension, size of the aneurysm and levels of matrix metalloproteinases-9 and C-reactive protein. DESIGN, SETTING, SUBJECTS: This case-control study was designed to quantify the circulating levels of soluble co-stimulatory molecules by an in-house enzyme linked immunosorbent assay. A total of 314 subjects participated in the study including 100 patients and 214 normal controls. The statistical analysis was performed by Mann-Whitney test and Spearman's correlation rank test. RESULTS: Our results show increased plasma levels of sCD28, sCD86 (P = 0.0001) and decreased plasma levels of sCTLA-4 (P = 0.0018) in the patients compared with normal individuals. The levels of these factors were not related to the age of the patient, size of aneurysm or levels of C-reactive protein in plasma. There was, however, a significant inverse relationship between the concentrations of sCTLA-4 and sCD80 with matrix metalloproteinase-9. CONCLUSIONS: We suggest that soluble co-stimulatory molecules serve as biomarkers for the estimation of immune activation in AAA patients.

Basic and translational neonatal neuroscience research: whither goest the future of physician-scientists?

OBJECTIVES: In light of declining numbers of physician-scientists, the goal of this project was to identify strategies to invigorate and attract new talent to clinical research in the field of pediatric neurosciences. DESIGN: To develop a broad perspective, a program of direct questions was addressed to both US and non-US physicians at all stages of career development. RESULTS: Respondents identified numerous promising avenues of research but also indicated obstacles to research progress at all stages of career development including medical students, resident physicians, junior medical faculty, mid-career faculty, and senior faculty. At each career stage, ideas were offered to attract resources for, build prestige for, and motivate commitment for participation in clinical research. CONCLUSIONS: Creative promotion of clinical research at all stages of medical education and career development offers great promise to expand current physician-scientist numbers, and thereby stimulate many exciting advances in medicine.

Hematopoietic stem cell transplantation for cardiac and peripheral vascular disease.

Recent studies have suggested that marrow and blood hematopoietic stem cells may contribute to nonhematopoietic tissue repair in multiple organ systems. In animal models and more recently in limited human trials, unpurified marrow mononuclear cells and/or subsets of adult hematopoietic stem cells have been reported to contribute to neoangiogenesis. Since the subset of hematopoietic stem cells (HSCs) that are both CD34+ and AC133+ are endothelial cell precursors, clinical trials using autologous AC133+ HSCs isolated with the Miltenyi CLIMACS cell separator and transplanted into patients with ischemic and refractory peripheral vascular or coronary artery disease are being implemented at Northwestern University.

ERK inhibition attenuates 5-HT-induced contractions in fetal and adult ovine carotid arteries.

Growth and differentiation-related pathways are much more active in immature than in mature, fully differentiated smooth muscle. Because mitogen-activated protein kinases (MAPK) are intimately involved with growth and differentiation, and the extracellular signal-regulated kinase (ERK) subfamily of MAPKs are involved in some contractile responses, the present studies examined the hypothesis that ERKs play an important and age-dependent role in smooth muscle contraction. The MAPK inhibitors PD098059 and UO126 both inhibited serotonin (5-HT) concentration-response relations more effectively in carotid arteries from term fetal lambs, than in corresponding arteries from mature non-pregnant adult sheep. This inhibition involved significant decreases in both the pD2 (adult: 2-fold; fetus: 4- to 15-fold) and the maximum efficacy (adult: 15-19%; fetus: 34-39%) of 5-HT. Accompanying this age-dependent effect on contraction, quantitative Western blot assays revealed that ERK1 and ERK2 abundances were 39% and 164% greater, respectively, in fetal than in adult carotid arteries. The abundance of the putative ERK target, caldesmon, however, was about 7-fold greater in adult than in fetal arteries. Together, the present results support the view that ERK abundance and activity is upregulated in fetal relative to adult arteries, and that one consequence of this upregulation is that the contribution of ERKs to contraction, at least that initiated by 5-HT2a receptors, is greater in fetal than adult carotid arteries. Whereas the phosphorylation mechanisms through which ERKs augment contraction remain uncertain and controversial, the present results suggest that emphasis should be shifted away from caldesmon and toward other critical contractile proteins, and how these proteins may contribute differently to development of agonist-induced contractile force in immature and mature arteries.

Morphometric analysis of the popliteal artery for endovascular treatment.

This study intended to determine the precise diameter of the popliteal artery in patients at risk for popliteal aneurysms. Accurate sizing is necessary to develop devices for endovascular treatment of popliteal aneurysms. Fifty-four patients with abdominal aortic aneurysms (AAAs) had computed tomography (CT) scans of the popliteal arteries. Age- and gender-matched control subjects were measured by ultrasound. NIH Image was used to measure the minor diameter at the adductor hiatus (proximal) and femoral condyles (midpopliteal artery). There were 4 unsuspected popliteal aneurysms (7.4%). The proximal popliteal artery was ectatic in these patients: 13.4 +/- 5.2 mm. Proximal and midpopliteal arteries were significantly larger in the other patients with AAAs compared with controls: 9.6 +/- 1.8 mm vs 7.9 +/- 1.1 mm at the hiatus (p<0.001) and 10.2 +/- 2 mm vs 7.9 +/- 0.9 mm at the condyles (p<0.001). The popliteal artery was focally larger in patients with AAAs without popliteal aneurysms. The popliteal artery was larger in men compared with women; 9.8 +/- 1.8 mm vs 8.8 +/- 1.9 mm at the hiatus (p=0.024) and 10.5 +/- 1.9 mm vs 9.0 +/- 2.4 mm at the condyles (p=0.005). The proximal popliteal artery was 2 mm larger in patients at risk for popliteal aneurysms and 5 mm larger in patients with popliteal aneurysms compared to controls. Focal ectasia of the midpopliteal artery was common. Planning for endovascular treatment of popliteal aneurysms must incorporate this striking enlargement.

Antigen presentation by macrophages is enhanced by the uptake of necrotic, but not apoptotic, cells.

The aim of this study was to determine whether phagocytosis of necrotic or apoptotic cells affects antigen presentation by murine bone marrow-derived macrophages. After uptake of necrotic neutrophils, macrophages were able to stimulate significantly higher T cell proliferation in vitro against both the recall antigen albumin and the mitogen concanavalin A. No such effect was seen following phagocytosis of apoptotic neutrophils. Flow cytometry revealed that, within 4h of ingestion, macrophages that had taken up the necrotic cells expressed higher levels of CD40 than those that had phagocytosed apoptotic cells. Macrophage cultures pulsed with apoptotic, but not necrotic, neutrophils contained higher levels of transforming growth factor beta1, but lower concentrations of tumour necrosis factor alpha, compared to untreated controls. Our interpretation of these results is that macrophages that have taken up necrotic neutrophils co-stimulate T cells with greater efficiency due to rapid CD40 up-regulation, whereas those that have ingested apoptotic cells are not only ineffective in co-stimulation, but also secrete inhibitory cytokine.

Simultaneous glutamate and gamma-aminobutyric acid release within ventrolateral medulla during skeletal muscle contraction in intact and barodenervated rats.

The purpose of this study was to determine if baroreflex modulates cardiovascular responses and neurotransmitter release within rostral (RVLM) and caudal (CVLM) ventrolateral medulla during static contraction of skeletal muscle using anesthetized rats. We evoked cardiovascular responses by a static muscle contraction and measured simultaneous release of glutamate and gamma-aminobutyric acid (GABA) in both the RVLM and CVLM using microdialysis probes, two inserted bilaterally into the RVLM and two into the CVLM. In intact anesthetized rats, a muscle contraction increased release of glutamate concomitantly in both the RVLM and CVLM along with significant increases in heart rate and arterial blood pressure. In contrast, concentrations of GABA increased within the RVLM, but decreased significantly within the CVLM during the pressor response. These changes were due to contraction-evoked activation of muscle afferents since tibial nerve stimulation following muscle paralysis failed to evoke glutamate, GABA, or any cardiovascular changes. On the other hand, static muscle contractions in baroreceptor denervated rats augmented the increases in heart rate and blood pressure. Furthermore, muscle contraction significantly enhanced the release of glutamate in the RVLM but attenuated its release in the CVLM. In addition, concentrations of GABA within the RVLM were attenuated following a muscle contraction in denervated rats without any changes in GABA within the CVLM. These results demonstrate that the baroreceptors influence cardiovascular responses to static muscle contraction associated with dynamic changes in glutamate and GABA release within the RVLM and CVLM.

Modulation of pressor response to muscle contraction via monoamines following AMPA-receptor blockade in the ventrolateral medulla.

We hypothesized that cardiovascular responses to static muscle contraction are mediated via changes in extracellular concentrations of monoamines (norepinephrine, dopamine and serotonin) following the administration of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, an AMPA-receptor antagonist) into the rostral (RVLM) or caudal (CVLM) ventrolateral medulla. For the RVLM experiments (n= 8), a 2-min static muscle contraction increased the mean arterial pressure (MAP) and heart rate (HR) by 23 +/- 2 mmHg and 28 +/- 8 bpm, respectively. During this contraction, the concentrations of norepinephrine, dopamine, and serotonin within the RVLM increased by 278 +/- 52%, 213 +/- 23%, and 232 +/- 24%, respectively. Microdialysis of CNQX (1.0 microM) for 30 min into the RVLM attenuated the increases in MAP and HR ( 11 +/- 2 mmHg and 14 +/- 5 bpm) without a change in developed muscle tension. The levels of norepinephrine, dopamine, and serotonin within the RVLM were also attenuated. In contrast, microdialysis of CNQX into the CVLM (n= 8) potentiated the contraction-evoked responses in MAP ( 21 +/- 2 vs 33 +/- 5 mmHg) and HR ( 25 +/- 5 vs 46 +/- 8 bpm) without any effect on the monoamine levels within the CVLM region. These results suggest that AMPA-receptor blockade within the RVLM and CVLM has opposing effects on cardiovascular responses during static muscle contraction. In addition, such receptor blockade modulates extracellular concentrations of monoamines within the RVLM but not in the CVLM. These results provide evidence that AMPA receptors within the ventrolateral medulla play a role in exercise pressor reflex.

Effects of nitric oxide and GABA interaction within ventrolateral medulla on cardiovascular responses during static muscle contraction.

We hypothesized that nitric oxide (NO) has opposing roles in regulating cardiovascular responses within the rostral (RVLM) and caudal (CVLM) ventrolateral medulla by modulating release of gamma-aminobutyric acid (GABA). We have measured GABA concentrations within the RVLM and CVLM during increases in mean arterial pressure (MAP) and heart rate (HR) following a 2-min tibial nerve stimulation-evoked static muscle contraction before and after microdialysis of the NO precursor, L-arginine (1.0 microM), for 30 min, and after the NO inhibitor, L-NMMA (1.0 microM), for 30 min. In eight anesthetized rats, muscle contraction significantly increased MAP, HR and GABA levels within the RVLM area (from 0.53+/-0.09 to 1.22+/-0.10 ng/10 microl). Following microdialysis of L-arginine, muscle contraction augmented GABA levels (from 0.45+/-0.07 to 2.18+/-0.09 ng/10 microl) and attenuated changes in MAP and HR. Subsequent application of L-NMMA significantly decreased GABA levels (from 0.47+/-0.08 to 0.22+/-0.07 ng/10 microl) but potentiated MAP and HR responses to a muscle contraction. In contrast, muscle contraction significantly increased MAP and HR but decreased GABA concentrations within the CVLM (from 1.20+/-0.20 to 0.78+/-0.17 ng/10 microl). Following microdialysis of L-arginine, muscle contraction significantly attenuated GABA levels (from 1.34+/-0.19 to 0.33+/-0.10 ng/10 microl) and augmented changes in MAP and HR in response to muscle contraction. A subsequent microdialysis of L-NMMA into the CVLM reversed the effects of L-arginine. These results demonstrate that NO within the RVLM and CVLM differentially modulates cardiovascular responses during static muscle contraction and that NO influences exercise-induced cardiovascular responses by modulating GABA release within the ventrolateral medulla.

Animal models of neonatal stroke.

Neonatal stroke occurs in approximately 1 in 4,000 to 1 in 10,000 newborns, and more than 80% involve the vascular territory supplied by the middle cerebral artery. Neonatal stroke is associated with many acquired and genetic prothrombotic factors, and follow-up studies indicate that as many as two thirds of neonates develop neurologic deficits. In the past two decades unilateral carotid occlusion with 8% hypoxia has been used to study focal and global ischemia in the newborn, and recently a filament model of middle cerebral artery occlusion has been developed. This review describes the results of studies in these two newborn models covering aspects of the injury cascade that occurs after focal ischemia. A likely requirement is that therapeutic efforts be directed less at using thrombolytic therapy and more toward treatment of events associated with reperfusion injury, the inflammatory cascade, and apoptosis. Additional areas of research that have received attention in the past year include inhibition of nitric oxide and free-radical formation, use of iron chelating agents, the potential role of hypoxia-inducible factors and mediators of caspase activity, use of growth factors, hypothermia, and administration of magnesium sulfate.

Is bilateral ultrasound scanning of the legs necessary for patients with unilateral symptoms of deep vein thrombosis?

PURPOSE: The purpose of this study was to determine the necessity of bilateral lower-extremity venous duplex ultrasound scanning in patients with unilateral symptoms of deep vein thrombosis (DVT). PATIENTS AND METHODS: A retrospective review of 1080 bilateral venous duplex scans was performed. Patients were randomly selected from a total of 7922 studied between May 1998 and May 2000. Data on patient age, sex, comorbidity, and the reason for ultrasound scan were compiled. Forty percent (435/1080) of patients presented with unilateral symptoms of lower-extremity DVT. This group was further analyzed according to their status as inpatients or outpatients. RESULTS: DVT was diagnosed in 26.9% (117/435) of the patients. Of the inpatients found to have DVT, the thrombus was confined to the symptomatic leg in 23.8% (38/159), thrombus was present just in the asymptomatic leg in 8/159 (5.0%), and thrombus was found in both legs in 8/159 (5.0%). In the outpatient group, thrombus was confined to the symptomatic leg in 21.0% (58/276) and found in both legs in 1.8% (5/276). None of the 276 outpatients had DVT isolated in the asymptomatic leg. CONCLUSION: Routine bilateral lower-extremity venous duplex studies are not necessary in outpatients presenting with unilateral symptoms. In many outpatients, a single-limb study will suffice. If a patient is found to have a DVT on the symptomatic side, then we believe that a bilateral study is indicated. We do believe that routine bilateral scanning of inpatients remains justified. This algorithm may save technician time and increase vascular laboratory efficiency.

Developmental changes in ryanodine- and IP(3)-sensitive Ca(2+) pools in ovine basilar artery.

To explore the hypothesis that cerebrovascular maturation alters ryanodine- and inositol 1,4,5-trisphosphate (IP(3))-sensitive Ca(2+) pool sizes, we measured total intracellular Ca(2+) with (45)Ca and the fractions of intracellular Ca(2+) released by IP(3) and/or caffeine in furaptra-loaded permeabilized basilar arteries from nonpregnant adult and term fetal (139-141 days) sheep. Ca(2+) mass (nmol/mg dry weight) was similar in adult (1.60 +/- 0.18) and fetal (1.71 +/- 0.16) arteries in the pool sensitive to IP(3) alone but was significantly lower for adult (0.11 +/- 0.01) than for fetal (1.22 +/- 0.11) arteries in the pool sensitive to ryanodine alone. The pool sensitive to both ryanodine and IP(3) was also smaller in adult (0.14 +/- 0.01) than in fetal (0.85 +/- 0.08) arteries. Because the Ca(2+) fraction in the ryanodine-IP(3) pool was small in both adult (5 +/- 1%) and fetal (7 +/- 4%) arteries, the IP(3) and ryanodine pools appear to be separate in these arteries. However, the pool sensitive to neither IP(3) nor ryanodine was 10-fold smaller in adult (0.87 +/- 0.10) than in fetal (8.78 +/- 0.81) arteries, where it accounted for 72% of total intracellular membrane-bound Ca(2+). Thus, during basilar artery maturation, intracellular Ca(2+) mass plummets in noncontractile pools, decreases modestly in ryanodine-sensitive pools, and remains constant in IP(3)-sensitive pools. In addition, age-related increases in IP(3) efficacy must involve factors other than IP(3) pool size alone.