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Biomedical subjects

C Reilly

Publications and source records attributed to C Reilly.

At least 19 recordsLinked to original sources

Selenium levels in cow's milk from different regions of Australia.

Homogenised cow's milk samples were collected each season in 1990 and 1991 from 19 selected dairy factories in different regions across Australia. Fresh cow's milk samples were also collected every month in 1990 and 1991 from 24 selected dairy farms in Queensland. Selenium (Se) analyses were performed by spectrofluorimetry after complexing with 2,3-diaminonaphthalene. The study found a wide variation of Se levels in homogenised milk from dairy milk factories. Statistical analysis using unpaired t-test showed that Se levels in summer were significantly higher (P < 0.001) than in other seasons. Mean concentrations in summer, autumn, winter and spring for 1990 and 1991 were 23.8 +/- 4.6, 20.9 +/- 4.4, 20.7 +/- 4.2 and 20.6 +/- 4.8 micrograms/L, respectively. A two-way ANOVA analysis also indicated that regions and season have a significant influence (P < 0.001) on Se levels and that there is an interaction between regions and seasons. High Se levels in each region tend to be associated with high Se levels in summer milk. A wide variation of Se levels was also found in raw milk collected from dairy farms. Mean concentration was 22.0 +/- 6.0 micrograms/L, with a range of 6.7 to 47.6 micrograms/L. When the values of Se levels in homogenised milk for each season were used to estimate the contribution of milk to daily intake of different populations groups, it was found that infants have the highest intake (10.0-12.2 micrograms/day) which meets the Australian Recommended Dietary Intake.

Analysis of Variance↗

Duke's 3rd year: a 35-year retrospective.

PURPOSE: Our purpose was to review and evaluate Duke University School of Medicine's 3rd year. SUMMARY: Duke University School of Medicine instituted a dramatic change in its curriculum 35 years ago by restructuring the 1st, 2nd, and 4th years of medical school so that the entire 3rd year could be devoted to an elective research experience or independent study of the basic sciences. Today this "new curriculum" permits students to work in basic science laboratories or with clinical investigators, work toward a 2nd degree, and participate in basic science courses beyond the core curriculum. Students were surveyed in 1996 and 1998 about their 3rd-year experience. Results showed that 67% and 73%, respectively, believed the 3rd year was very helpful in facilitating their understanding and appreciation of biomedical and clinical research. Students especially valued the experience and availability of their preceptors and the range of opportunities available during the 3rd year. The most frequent critiques by students were the lack of opportunities to pursue clinical interests, difficulty integrating the 3rd year into the remainder of the curriculum, and a tendency to place too much emphasis on coupling the 3rd-year experience with scholarships and residency plans rather than on a specific investigative interest. CONCLUSIONS: Overall, both faculty and students value the 3rd year as a unique positive experience. Duke faculty have noted, however, that no other medical schools have attempted to replicate the Duke experience.

Curriculum↗

Extended scope of nursing practice: a multicentre randomised controlled trial of appropriately trained nurses and pre-registration house officers in pre-operative assessment in elective general surgery.

OBJECTIVES: (1) To determine whether pre-operative assessment carried out by an appropriately trained nurse (ATN) is equivalent in quality to that carried out by a pre-registration house officer (PRHO). (2) To assess whether pre-assessments carried out by ATNs and PRHOs are equivalent in terms of cost. (3) To determine whether assessments carried out by ATNs are acceptable to patients. (4) To investigate the quality of communication between senior medical staff and ATNs. DESIGN: The study design was principally a prospective randomised equivalence trial but was accompanied by additional qualitative assessment of patient and staff perceptions, and an economic evaluation. SETTING: The study was carried out at four NHS hospitals, three of which were teaching hospitals, in three NHS Trusts in Southampton, Sheffield and Doncaster. SUBJECTS: All patients attending at one site for assessment prior to general anaesthetic for elective general, vascular, urological or breast surgery were potentially included in the study. Of 1907 patients who were randomised, 1874 completed the study with a full evaluation. INTERVENTIONS: The intervention consisted of a pre-operative assessment carried out by either an ATN or a PRHO. Of the patients who completed the study with a full evaluation, 926 patients were randomised to the PRHO arm of the trial and 948 to the ATN arm. Three ATNs took part in the study, one from each centre, together with a total of 87 PRHOs. MAIN OUTCOME MEASURES: Immediately following the initial assessment of a patient by a PRHO or an ATN, one of a number of clinical research fellows, all specialist registrars in anaesthetics, repeated the assessment and recorded it on a study form, together with a list of investigations required. The clinical research fellow then evaluated the competency of the initial assessor by comparing the quality of their assessment with their own. Any deficiencies in ordering of investigations and referral to other specialities were met in order to maximise patient care. Three areas of ATN and PRHO performance were judged separately, history taking, examination and ordering of tests, and each was graded into one of four categories, the most important of which was under-assessment, which would possibly have affected peri-operative management. In the case of ordering of tests, it was possible to have both over- and under-assessed a patient on different tests. RESULTS: The pre-operative assessments carried out by the ATNs were essentially equivalent to those performed by the PRHOs in terms of under-assessment that might possibly have affected peri-operative management, although there was variation between the ATNs in terms of the quality of history taking. This may be related to the low number of patients seen at one study site. PRHOs ordered significantly more unnecessary tests than the ATNs. The substitution of ATNs for PRHOs was calculated to be cost neutral. The results of the qualitative assessment showed that the use of ATNs for pre-operative assessment was acceptable to patients; however, there was no evidence that communication between senior medical staff and those carrying out pre-operative assessments was improved by their introduction. CONCLUSIONS: This study demonstrated no reason to inhibit the development of fully nurse-led pre-operative assessment, provided that the nurses are appropriately trained and maintain sufficient workload to retain skills. CONCLUSIONS--IMPLICATIONS FOR THE HEALTH SERVICE: ATNs provide an acceptable and efficient alternative to PRHOs for the purposes of routine pre-operative assessment. Consideration will have to be given, however, to the positions of these nurses within the surgical team, and also to their career structure. CONCLUSIONS--RECOMMENDATIONS FOR FUTURE RESEARCH: Further research is needed in the following areas: (1) the extent and type of training needed for nurses undertaking the pre-operative assessment role; (2) the use, costs and benefits of routine pre-operative testing.

Clinical Competence↗

Differential effects of 15-deoxy-delta(12,14)-prostaglandin J2 and a peroxisome proliferator-activated receptor gamma agonist on macrophage activation.

Prostaglandin J2 metabolite 15-deoxy-delta(12,14)-prostaglandin J2 (15-PGJ2) appears to possess anti-inflammatory properties. Unlike other prostaglandins, it has no known plasma membrane receptor. Its effects have been thought to occur through activation of the nuclear peroxisome proliferator-activated receptor gamma (PPARgamma), but 15-PGJ2 may exhibit effects independent of PPARgamma. We hypothesized that 15-PGJ2 modulates macrophage (Mphi) mediator production by acting on cell signaling proteins upstream of PPARgamma. The effects of 15-PGJ2 on bacterial endotoxin LPS-induced rat peritoneal Mphi mediator production were compared with those of a specific PPARgamma agonist, BRL 49653 (BRL), and to the eicosanoids prostaglandin D2 (PGD2) and cicaprost (CICA, a prostacyclin analogue). 15-PGJ2 inhibited LPS-induced production of NO, TNF-alpha, and thromboxane B2 (TxB2). Equimolar concentrations of PGD2 and CICA significantly inhibited LPS-stimulated TNF-alpha but not NO, and CICA increased TxB2 production. BRL inhibited LPS-induced NO, but augmented LPS-induced TNF-alpha and TxB2. 15-PGJ2 also inhibited degradation of LPS-induced IkappaB alpha and phosphoactivation of ERK 1/2, but BRL had no significant effect on either protein. The cyclopentenone ring 2-cyclopenten-1-one also inhibited LPS-induced ERK 1/2 activation; however, neither 15-PGJ2 nor the cyclopentenone inhibited PMA-induced ERK 1/2 activation. Inhibition of LPS-stimulated mediator production by 15-PGJ2 differed from inhibition by PGD2, CICA, and BRL. The ability of 15-PGJ2 to inhibit LPS-induced Mphi mediator production and cell signaling may occur in part through reactivity of its cyclopentenone ring.

Animals↗

Transgenic expression of Ly-49A in thymocytes alters repertoire selection.

A T cell-specific Ly-49A transgene inhibits TCR-mediated activation in the presence of H-2Dd. Expression of this transgene by developing thymocytes impairs negative selection evidenced by a failure to delete potentially autoreactive T cells and development of a graft-vs-host-disease-like syndrome. In mice carrying both the Ly-49A and a class II-restricted TCR transgene, positive selection was lost, but only when H-2Dd was present on thymic epithelium. These results are consistent with models suggesting that thymic selection is dependent on the perceived intensity of TCR signaling. More interestingly, these results show that Ly-49A does not simply provide a strict on/off switch for T cell responses. Since Ly-49A may shift the signaling threshold of TCR-induced triggering, inducible expression of Ly-49A may regulate peripheral memory/activated T cells by raising the threshold for T cell reactivation.

Animals↗

Potassium-magnesium citrate versus potassium chloride in thiazide-induced hypokalemia.

BACKGROUND: The purpose of this study was to compare the value of potassium-magnesium citrate (KMgCit) with potassium chloride in overcoming thiazide-induced hypokalemia. METHODS: Sixty normal subjects first took hydrochlorothiazide (HCTZ; 50 mg/day). After three weeks of treatment (or earlier if hypokalemia developed), they were randomized to take KMgCit (42 mEq K, 21 mEq Mg, and 63 mEq citrate/day) or potassium chloride (42 mEq/day) for three weeks while continuing on HCTZ. RESULTS: KMgCit significantly increased the serum potassium concentration from 3.42 +/- 0.30 mEq/L on HCTZ alone to about 3.8 mEq/L (P < 0.001). Potassium chloride produced a similar increase in serum potassium concentration from 3.45 +/- 0.44 mEq/L to about 3.8 mEq/L (P < 0. 001). KMgCit significantly increased the serum magnesium concentration by 0.11 to 0.12 mEq/L (P < 0.01), whereas potassium chloride produced a marginal decline or no significant change. KMgCit was less effective than potassium chloride in correcting HCTZ-induced hypochloridemia and hyperbicarbonatemia. KMgCit, but not potassium chloride, significantly increased urinary pH (by about 0.6 unit), citrate (by about 260 mg/day), and urinary magnesium. CONCLUSIONS: KMgCit is equally effective as potassium chloride in correcting thiazide-induced hypokalemia. In addition, KMgCit, but not potassium chloride, produces a small but significant increase in serum magnesium concentration by delivering a magnesium load, and it confers alkalinizing and citraturic actions.

Adult↗

Technical aspects of trace element supplementation.

Routine supplementation of total parenteral nutrition mixtures with the readily available single or combination trace elements products is becoming more widespread. As more is learned about deficiency syndromes and monitoring techniques, so too must we understand more about the physicochemical interactions between individual trace elements and other nutrients, that could ultimately affect bioavailability. Expert pharmaceutical assessment of these complex reactions, that have been demonstrated to occur in solution, becomes increasingly important in order to optimize the efficacy of micronutrient therapy.

Dietary Supplements↗

Correlation of serum measures of nitric oxide production with lupus disease activity.

OBJECTIVE: To determine whether serum measures of nitric oxide production correlate with disease activity in patients with systemic lupus erythematosus (SLE). METHODS: We assayed the levels of serum nitrate/nitrite from 26 patients with SLE followed for 1-3 years and nitrotyrosine levels in sera from 28 additional patients with SLE; sera from 19 controls were tested in both assays. Lupus disease activity was determined via the physician's global assessment, the Lupus Activity Index, and the SLE Disease Activity Index (SLEDAI) at the time of serum collection for the initial set of 26 patients. Statistical correlations were determined using the Wilcoxon rank sum method and one-way ANOVA testing. RESULTS: Serum levels of nitrate/nitrite were significantly higher in 26 patients with SLE compared to 19 controls (SLE, mean 29.5 microM/ml, range 1-438; controls, mean 9.6 microM/ml, range 0-51; p = 0.0004). Overall, there was a significant correlation between serum nitrate/nitrite levels and SLEDAI scores (p = 0.0065). Renal variables within the SLEDAI had the highest correlation with serum nitrate/nitrite (p = 0.0028). Serum nitrotyrosine levels were also significantly higher in patients with SLE versus controls (p = 0.007) and in active SLE versus those with inactive SLE (p = 0.008). CONCLUSION: Serum nitrate/nitrite levels correlated with SLE disease activity, especially nephritis, in the majority of patients studied. Serum nitrotyrosine levels also differentiated controls from patients with lupus and patients with active from those with inactive disease. Due to the ease and low cost of these assays, serum measures of nitric oxide production appear a potentially useful adjunctive laboratory measure of disease activity in SLE and further implicate nitric oxide as an important mediator of disease in SLE.

Adult↗

Thymic skewing of the CD4/CD8 ratio maps with the T-cell receptor alpha-chain locus.

The thymic preference for CD4+ T cells over CD8+ T cells is often attributed to a default pathway favouring CD4+ T cells or to homeostatic mechanisms. It is also clear, however, that T-cell receptor (TCR) preferences for major histocompatibility complex (MHC) class I versus class II binding will strongly influence an individual clone's skewing to the CD4 or CD8 subset. The variable region of each TCR alpha chain (V alpha) studied to date is found to be overrepresented in either CD4+ or CD8+ cells, suggesting that each V alpha element can interact more favourably with either MHC class I or class II molecules. Indeed, TCRs appear to have an intrinsic ability to interact with MHC molecules, and single amino acid residues present in germline-encoded complementarity determining region 1 (CDR1) and CDR2 of the V alpha element can be responsible for determining MHC specificity. Interestingly, the degree of CD4/CD8 skewing is variable among different mouse strains and in human populations. Here, we have shown that polymorphism in CD4/CD8 skewing between B6 and BALB/c mice is determined by the stem cell genotype and not by environmental effects, and that it maps in or near the TCR alpha-chain complex, Tcra. This was confirmed by comparing Tcra(b) with Tcra(a) or Tcra(c) haplotypes in congenic mice. We propose that the array of V alpha genes in various Tcra haplotypes exerts influence over the proportion of CD4 and CD8 subsets generated and may account in part for the observed thymic skewing. Thus, while it has been suggested that the TCR genes have been selected by evolution for MHC binding, our results further indicate selection for class II MHC preference.

Animals↗

The density of the class II MHC T cell receptor ligand influences IFN-gamma/IL-4 ratios in immune responses in vivo.

Activation of CD4+ T cells depends on T cell receptor recognition of MHC class II/peptide and on costimulation provided by CD28/B7. It has been shown that different levels of costimulation can influence T helper cell differentiation into Th1 versus Th2 phenotypes. Similar arguments have been made for different levels of peptide/MHC density on antigen-presenting cells, but to date supportive evidence has only come from in vitro studies. Here, using transgenic mice with reduced MHC class II expression on both B cells and dendritic cells, we demonstrate that T helper cell differentiation in vivo is also influenced by the density of expression of MHC class II. Although priming and expansion of antigen-specific T cells were normal in these mice, T cell responses were dominated by the Th1-associated cytokine IFN-gamma, with reduced levels of the Th2 cytokine IL-4 compared to controls. These results provide direct evidence that the efficiency of antigen presentation in vivo can determine effector cell phenotype.

Adjuvants, Immunologic↗

The oestrogen receptor regulates NFkappaB and AP-1 activity in a cell-specific manner.

Oestrogens regulate the expression of genes both positively and negatively in a range of cell types. These effects are mediated via the oestrogen receptor (ER) and involve direct interactions between the ER and DNA response elements, as well as interactions between the ER and other nuclear proteins. We have examined the potential of the ERalpha to regulate the expression of reporter genes under the control of oestrogen response elements (EREs), NFkappaB response elements (NREs) or AP-1/TPA response elements (TREs) in HeLa cells and in human embryonic kidney (HEK-293) cells. Transiently transfected ERalpha was able to activate expression of beta-galactosidase under the control of EREs in an oestradiol (E2)-dependent manner in both HeLa and HEK-293 cells. The ERalpha was able to repress by 80% the TNF-mediated expression of beta-galactosidase under the control of NREs in an E2-dependent manner in HeLa cells but not in HEK-293 cells. ERalpha/E2 also induced a two-fold potentiation of TPA-mediated expression of beta-galactosidase under the control of TREs in HeLa cells but not in HEK-293 cells. These results suggest that the ERalpha is capable of regulating gene expression in a cell-specific manner. We further investigated the mechanisms by which the ERalpha regulates gene expression in these systems by co-expressing the ERalpha and the reporter gene constructs with known cofactors of the ERalpha. We have shown that expression of steroid receptor coactivator-1 alpha (SRC-1alpha) and receptor interacting protein-140 (RIP-140) have no effect on the capacity of the ERalpha to modulate NFkappaB reporter gene activity in HeLa cells. Furthermore, the expression of SRC-1alpha or RIP-140 does not enable the ERalpha to repress NFkappaB or to potentiate an AP-1 response in HEK-293 cells. This suggests that factors other than SRC-1alpha or RIP-140 are responsible for the cell-specific effects seen with ERalpha.

Dexamethasone↗

Transient improvement in glycemic control. The impact of pregnancy in women with IDDM.

OBJECTIVE: Good glycemic control throughout pregnancy in patients with diabetes is of paramount importance but often appears to deteriorate in the postpartum period. The aim of this study was to ascertain the timing of the improvement in glycemic control associated with pregnancy in women with IDDM and to examine changes in glycemic control after delivery. RESEARCH DESIGN AND METHODS: Peripartum glycemic control was assessed in a retrospective study of 30 women with IDDM whose age was 28 +/- 6 years (means +/- SD) and whose diabetes duration was 14 +/- 6 years. RESULTS: Mean total HbA1 fell incrementally from a peak at 2-3 years preconception to a nadir between 24 weeks and term, only to return to preconception levels within a year after delivery. Of the 30 women, 15 (50%) attained an HbA1 in the nondiabetic range for pregnancy at some point during their pregnancy, and 7 (23%) women achieved this by 24 weeks gestation. Women with an HbA1 > 9% at booking had a significantly higher HbA1 at 0-6 and 6-12 months preconception, and throughout pregnancy their HbA1 was significantly higher. After delivery, attendance rates at routine diabetes review clinics were low, with 11% of women not attending for longer than 24 months. CONCLUSIONS: Nearly all women with IDDM can achieve near normoglycemia during pregnancy, irrespective of previous glycemic control, although those with high HbA1 levels at booking are less likely to achieve this. After delivery, glycemic control deteriorates. Efforts to improve glycemic control should be intensified in the preconception period and maintained after delivery. The poor postpartum attendance at diabetes clinics requires specific action.

Adult↗

Identification of a new mouse beta-chemokine, thymus-derived chemotactic agent 4, with activity on T lymphocytes and mesangial cells.

Thymus-derived chemotactic agent 4 (TCA4), a new member of the beta-chemokine family, was cloned from a mouse thymic cDNA library. High levels of TCA4 mRNA are expressed in thymus; lower levels of message are found in spleen, heart, and kidney. Anti-TCA4 antibodies were used to localize sites of TCA4 expression within lymphoid tissues. In the thymus, UEA-1+ medullary epithelial cells, some endothelial cells, and additional undefined stromal elements were stained with anti-TCA4. TCA4 was also expressed as a meshlike network in splenic white pulp and in the medullary region of the lymph nodes. In addition, some lymph node and splenic blood vessels stained with anti-TCA4 antibodies. Rel B NFkappaB-deficient mice lack a transcription factor required for the generation of dendritic cells and the development of an organized thymic medulla. Rel B-deficient animals express very low levels of TCA4 in the thymus and little or no TCA4 in the periphery. At subnanomolar concentrations, TCA4 is a chemoattractant of mature T cells; the potential role of this novel chemokine in facilitating normal lymphocyte traffic is discussed. TCA4 is also a chemoattractant of cultured mesangial cells. Neutralizing anti-TCA4 mAb was used to demonstrate the specificity of TCA4-mediated cell migration. Finally, competitive binding studies with a SV40-transformed mouse mesangial cell line demonstrated that other murine beta-chemokines (monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, and thymus-derived chemotactic agent 3) do not compete for TCA4 binding.

Amino Acid Sequence↗

Thymic cortical epithelium is sufficient for the development of mature T cells in relB-deficient mice.

Thymic development of T lymphocytes progresses as a consequence of both TCR-mediated and non-TCR-mediated interactions between thymocytes and stromal cells. As relB-deficient mice appear to lack thymic medullary epithelium and mature dendritic cells, we studied the effect of this "cortex-only" thymus on T cell development. Two major consequences were observed. First, in both relB mutant and TCR transgenic/relB mutant mice, positive selection of both TCR alpha beta and delta gamma T cells appeared to proceed normally, with export of fully functional T cells to the periphery, suggesting that the thymic medullary stromal cells are not required for full maturation of T cells nor is an organized medullary compartment required for accumulation of mature single positive CD4 and CD8 T cells. Second, thymic negative selection was impaired, as evidenced by significant autoreactive proliferative responses to normal spleen stimulators. Peripheral T cells in relB mutant mice showed an unusually high proportion of CD69+ and CD44high cells. While some of these cells may be autoreactive T cells, most of the cells appeared to be activated by cytokines produced by relB mutant nonlymphoid cells, as the effect is minimized in relB mutant bone marrow chimeras. In sum, while the TCR-mediated steps in T cell maturation require both thymic cortex and medulla (epithelium and dendritic cells) for normal positive and negative selection of the repertoire, non-TCR-mediated interactions in the thymic cortex alone are sufficient to generate mature functional T cells.

Animals↗

Dendritic-like cells from relB mutant mice.

Mice deficient in the NF-kappa B transcription factor relB appear to have defects in the production of mature dendritic cells, as secondary lymphoid tissues are absent, and spleen cells show a significant loss of antigen presenting function. Moreover, the thymus appears to be impaired in negative selection, and immune responses in vivo are poor. Since dendritic cell precursors such as skin Langerhans cells appear to be normal, we sought information on the nature of the dendritic cell defect in these mice. Cultures of mutant bone marrow in the presence of GM-CSF revealed a delay in the accumulation of cells with dendritic cell features relative to controls; however, these cells were nearly as potent on a per cell basis as wild type cells in the stimulation of allogeneic mixed lymphocyte cultures. Similarly, skin Langerhans cells from mutant mice also showed significant ability to stimulate allogeneic T cells in culture. Since these findings cannot explain the defect in immune responses and the absence of secondary lymphoid tissues, we also looked at the ability of the relB mutant dendritic-like cells to form aggregates in vitro with naive syngeneic T cells. In this case, while wild type dendritic cells generated compact aggregates with T cells, relB mutant cells only formed irregular small aggregates. Thus, while relB mutant dendritic-like cells have some functions of mature dendritic cells, other functions are deficient. Understanding the role of relB in regulation of these functions should lead to a greater understanding of the molecular basis of dendritic cell development and function.

Animals↗

Mild renal pelvic dilatation is not predictive of vesicoureteral reflux in children.

OBJECTIVE: To determine if mild renal pelvic dilatation at renal ultrasound (RUS) is a reliable sign of vesicoureteral reflux (VUR) at voiding cystourethrogram (VCUG) in children. MATERIALS AND METHODS: All patients less than 10 years of age who had RUS and VCUG on the same day during a 2-year period were identified in a computerized database. The appearance of the collecting system of each kidney was classified into two groups: group 0 - no dilatation (</= 2-mm anteroposterior diameter of the renal pelvis) and group 1 - 3 to 10-mm AP diameter of the renal pelvis without caliectasis. VUR at VCUG was graded using the International Reflux Study Committee system. RESULTS: Four hundred fifty-five patients (76 boys; 379 girls) with 910 kidneys were included. VUR occurred in 268 kidneys in 174 patients. There were 820 group 0 kidneys and 90 group 1 kidneys. Kidneys classified as group 1 (25.0 % had reflux) were no more likely to have reflux than were kidneys with nondistended (group 0) collecting systems (31.2 % had reflux). There was no statistical difference in the rate of reflux in patients with group 1 renal pelvic distention (39.2 % refluxed) and a normal collecting system (33.3 % refluxed) (P = 0.365). CONCLUSION: The frequency of vesicoureteral reflux in children with mild renal pelvic distention is not significantly different than in children with no distention. Therefore, mild dilatation of the renal pelvis should not be considered an indication for voiding cystourethrography.

Child↗

Monocyte chemoattractant protein-1: receptor interactions and calcium signaling mechanisms.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the Cys-Cys chemokine family. Two related MCP-1 receptors have been identified (CC-CKR2A and CC-CKR2B), although the precise kinetics of ligand binding and calcium signaling of these receptors has yet to be investigated. To examine this more closely, the human MCP-1 receptors were cloned and expressed in Chinese hamster ovary (CHO) cells. Membranes prepared from cells expressing CC-CKR2B bind MCP-1 selectively and with high affinity (Kd = 120 pM). MCP-1 stimulation of recombinant CHO cells expressing CC-CKR2B induces a rapid increase in intracellular Ca2+ through both receptor-operated Ca2+ channels and mobilization of Ca2+ from intracellular stores, and leads to a rapid temperature-dependent internalization of the ligand/receptor complexes. In contrast, recombinant CHO cells expressing CC-CKR2A, and membranes prepared from these cells, fail to bind detectable levels of MCP-1. However, MCP-1 stimulation of cells expressing CC-CKR2A induces a small but significant increase in intracellular Ca2+. Repeated stimulation of these cells with MCP-1 leads to a potentiation of the response to a level comparable to that seen in cells expressing CC-CKR2B. These observations suggest that the levels of cell surface CC-CKR2A are controlled by novel mechanisms.

Animals↗