Ventilatory patterns during hypoxia, hypercapnia, and exercise in adolescents with mild scoliosis.

Adolescents with mild, asymptomatic scoliosis (thoracic curvature less than 35 degrees) may have little or no impairment of resting lung volumes. Progression to more severe disease may, however, be accompanied by lung restriction, impaired exercise tolerance, and respiratory failure with CO2 retention. We wished to see whether adolescents with mild scoliosis and minimally abnormal resting pulmonary mechanics had impairment of their responses to hypercapnia, hypoxia, and progressive cycle exercise. Forty-four adolescents with idiopathic scoliosis were studied. The mean forced vital capacity (FVC), expressed as a percentage of the predicted value, was 94.3 +/- 2.2 (SE). The mean ventilatory response to hypercapnia (2.57 +/- 0.24 L/min/mm Hg) was within the normal range but was achieved with a tidal volume response (1.87 +/- .17% vital capacity [VC]/mm Hg) that was significantly lower than that previously reported in healthy young adults. Ventilatory responses to exercise were also within the normal range, the mean dyspnea index (VE-max/maximal voluntary ventilation) = 0.92 +/- 0.04. However, at a ventilation of 30 L/min, the tidal volume was 0.38 +/- 0.01% FVC, which was considerably lower than predicted. The tidal volume response to hypoxia was also abnormally low, the mean response being 0.52 +/- 0.059% VC/% decrease in arterial O2 saturation. These findings indicated that, even when scoliosis is asymptomatic and associated with minimal impairment of resting pulmonary function, abnormal patterns of ventilation occur during exercise or in response to chemical stimuli.

Abortive infection in HeLaCD4 cells by a primary HIV type 1 isolate: implications for differential host cell tropism.

The emergence of T cell-tropic, syncytium-inducing (T-tropic/SI) HIV-1 variants from the background of macrophage-tropic, non-syncytium-inducing (M-tropic/NSI) strains is associated with disease progression in infected individuals. HIV89.6 is a primary isolate with a transitional phenotype: like M-tropic strains it replicates in primary macrophages and lymphocytes but not in most transformed cells, yet it is also syncytium inducing. We have shown that HIV89.6 can utilize both the M-tropic and T-tropic cofactors CCR-5 and CXCR-4, respectively, in conjunction with CD4 for fusion and entry into otherwise nonpermissive nonhuman cells. To better understand the nature of restricted HIV89.6 infection of transformed cells, we analyzed its interaction with CD4-expressing transformed human HeLaCD4-LTR/beta-Gal cells, which contain the beta-galactosidase gene linked to the HIV-1 LTR. Here we show that HIV89.6 enters these cells and undergoes reverse transcription and integration. Furthermore, HIV89.6 induces LTR-driven beta-galactosidase expression, indicating Tat-dependent trans-activation, in a similar number of cells as the permissive T-tropic/SI isolate HIV(HXB). Acute infection with HIV89.6, however, produces markedly lower levels of p24 antigen and infectious virus per trans-activation-positive cell than HIV(HXB). In contrast, transfection results in high levels of expression for both viruses but HIV89.6 still fails to establish spreading infection. HIV89.6 is also blocked after entry in two other nonpermissive cell lines, SUP-T1 and U937. HIV89.6 arrest in HeLaCD4-LTR/beta-Gal cells at a stage subsequent to entry, reverse transcription, integration, and Tat expression is a novel level at which HIV-1 strain- and cell-specific restrictions define host cell tropism. These studies emphasize that complex patterns of tropism are determined by the interplay of permissive or restricted virus-cell interactions at multiple steps in the replication cycle.

Maximal inspiratory and expiratory pressures in adolescents. Normal values.

The measurement of maximal inspiratory and expiratory pressures at the mouth (MIP and MEP, respectively) provides a noninvasive clinical method for evaluating the strength of respiratory muscles. In an attempt to reconcile the widely divergent normal values reported in the literature for healthy adolescents, we have measured, using simple manometry, MIP and MEP in 112 white subjects, 76 adolescents and 36 healthy adults. For female adolescents the values for MIP and MEP were 76 +/- 25 and 86 +/- 22 cm H2O, respectively, and were significantly less than those for male adolescents (p less than 0.01), whose mean values were 107 +/- 26 and 114 +/- 35 cm H2O, respectively. Mean values for adolescents were comparable to values measured in adult control subjects, and for both adolescents and adults, mean values approximated the lower end of the previously reported ranges of normal values in healthy subjects. Thus, MIP and MEP in healthy adolescents are significantly greater in male subjects than female subjects, but are comparable to those of healthy adults of the same sex. Furthermore, these studies suggest that the choice of normal values for MIP and MEP must take into account significant methodologic differences among laboratories.

Initial clinical experience with the Ahmed Glaucoma Valve implant in pediatric patients.

OBJECTIVE: To evaluate the Ahmed Glaucoma Valve implant, an aqueous shunting device with a unidirectional valve mechanism, in patients younger than 18 years. DESIGN: Prospective case series. SETTING: Tertiary care hospital. PATIENTS: Twenty-one consecutive patients younger than 18 years. The median age of patients was 4.8 years (range, 0.23-17.9 years) INTERVENTION: Placement of an Ahmed Glaucoma Valve implant between April 1992 and April 1994. MAIN OUTCOME MEASURE: Time after surgery without failure. Success was defined as an average intraocular pressure less than 22 mm Hg for the last 2 follow-ups in eyes with preoperative intraocular pressure greater than 22 mm Hg, or an intraocular pressure that was lowered by at least 20% from preoperative values in eyes with preoperative intraocular pressure less than 22 mm Hg, and no additional glaucoma surgeries or visually devastating complications. RESULTS: Cumulative probabilities of success at 12 and 24 months were 77.9% +/- 8.8% and 60.6% +/- 13.7%, respectively. One eye had a flat anterior chamber and suprachoroidal hemorrhage on the first postoperative day. No other eyes had flat or shallow anterior chambers that required reformation. In 3 eyes the implant extruded from underneath the conjunctiva and was removed. In 2 other eyes the average intraocular pressure for the last 2 follow-ups was greater than 22 mm Hg. In 1 eye with an intraocular pressure less than 22 mm Hg preoperatively, the intraocular pressure was not reduced by at least 20%, although the number of antiglaucoma medications was reduced. CONCLUSION: The 12-and 24-month success rates of the Ahmed Glaucoma Valve implant are similar to those of other implants when used in a pediatric population.

Ear oximetry during combined hypoxia and exercise.

Ear oximetry is widely used to detect arterial O2 desaturation during exercise in patients with cardiopulmonary disease. Although oximeters have been evaluated for accuracy, response time, and the influence of skin pigmentation, tests of their reliability have not been reported during strenuous exercise. Accordingly, we compared arterial O2 saturation (Sao2) measurements obtained by Hewlett-Packard (HP, model 47201A) and Biox II oximeters with those determined directly from arterial blood in six healthy volunteers during progressive exercise while rebreathing hypoxic gas mixtures. The relationship between the HP oximeter value and blood Sao2 was described by the equation: HP = 0.93 (Sao2) + 5.37 and for the Biox II: Biox = 0.55 (Sao2) + 38.97. With these equations, at a blood Sao2 value of 90%, the underestimation by both oximeters was less than 2%. At a blood value of 70%, the HP oximeter overestimated blood Sao2 by 0.7%, whereas the Biox II showed an overestimation of 10.7%. Below blood Sao2 of 83%, the Biox II tended to overestimate blood Sao2 by an amount greater than the error of the instrument, whereas the HP estimations were within the error of the instrument over all levels of blood Sao2 studied. We conclude that the HP oximeter provides valid estimates of Sao2 during exercise but that the Biox II oximeter, although reflecting qualitative changes in oxygenation that occur during exercise, does not provide accurate records of the degree of desaturation.

Growth of rabbit pigmented and nonpigmented ciliary body epithelium.

Pigmented (PE) and nonpigmented epithelial (NPE) cells were carefully dissected from the ciliary processes of rabbit eyes and maintained in vitro. Both epithelial cultured cells showed hexagonal morphology by light microscopy; abundant granules containing pigment could be seen in PE cells while no pigment was seen in the NPE cells. S-100 protein exists in both cultured cells and was used as a marker for the in vitro confirmation of these cells. Collagen type III was used as a negative marker to rule out stromal cell contamination in tissue cultures. PE cells attached and proliferated well in 20% fetal bovine serum (FBS) in minimum essential media (MEM). Greater proliferation was attained when PE cells were exposed to 20% vitreous extract in addition to 20% FBS-MEM. Media conditioned by PE cells did not significantly stimulate PE cells. NPE cells showed greatest proliferation in 20% FBS-MEM with either 20% concentration of media conditioned by PE cells or when exposed to 20% concentration of vitreous extract. Rabbit vitreous may contain factor(s) which stimulate the proliferation of both cultured cells, and conditioned media from the PE cells may contain factor(s) which stimulate NPE cell proliferation.

Decreased levels of muscarinic receptors in bladders from the alcohol preferring rat line.

The Bmax for [3H]QNB binding in the bladders of alcohol preferring (AA) rats was only approximately 60% of that in the alcohol non-preferring (ANA) rats. No significant change in Bmax for [3H]QNB binding in bladder was observed between alcohol insensitive (AT) and alcohol sensitive (ANT) rats. No significant change in Kd for [3H]QNB binding in bladder was observed between the four different rat lines studied. Therefore, alcohol preference but not sensitivity is associated with a decrease in muscarinic receptor density in the rat bladder. Because all of the rats used in this study were ethanol-naive, the decrease in muscarinic receptor density in the bladders of alcohol preferring rats is associated with genetic factors inherent to this rat line. Further studies are needed to determine if these observations are tissue specific or specific to the m2 subtype, which predominates in the rat bladder.

Validation of noninvasive maximal cardiac output measurement.

Due to the invasiveness of direct techniques and problems that constrain the use of popular indirect techniques during very heavy (non-steady-state) exercise, measurements of maximal cardiac output are seldom included in studies of exercise. The acetylene-rebreathing technique is well-suited for maximal exercise; however, until recent technological advances, difficulties involved in collecting and measuring alveolar acetylene samples have restricted its use. We compared cardiac output values measured via the acetylene-rebreathing technique (QA) (modified for use with a mass spectrometer) and the dye-dilution technique (QD) at rest and during light to maximal exercise in six moderately active males. Although QA consistently underestimated QD, the two techniques showed a significant correlation of 0.87 throughout all levels of exercise. During maximal exercise, QA and QD values were not significantly different (24.7 and 26.7 l X min-1, respectively). Modifications in the QA technique that reduce potential sources of error were also examined. We conclude that the acetylene-rebreathing technique, modified for use with a mass spectrometer, is a simple and valid procedure for measuring maximal cardiac output.

Characterization of human and rabbit pigmented and nonpigmented ciliary body epithelium.

Nonpigmented epithelial (NPE) and pigmented epithelial (PE) cells were carefully dissected from both human and rabbit ciliary processes and have been maintained in vitro and partially characterized by morphology and immunocytochemical techniques using polyclonal and monoclonal antibodies against S-100 proteins, collagen type I and type III. The tissue distribution of these proteins was studied in formalin fixed deparaffinized tissue sections of human and rabbit eyes by immunoperoxidase staining techniques. Both NPE and PE cell lines from human and rabbit showed hexagonal morphology by light microscopy; distinct granules containing pigment could be visualized in the PE cell lines, but not in the NPE cells. Antibodies against S-100 proteins stained NPE layer intensely and PE layer slightly in the human tissue sections. The staining was less intense in rabbit tissues than human tissues. The ciliary body stroma was positive for collagen type III and negative for collagen type I or S-100.

Ventilatory care in status asthmaticus.

Asthma continues to pose a significant medical problem in terms of both morbidity and mortality. A number of patients with a severe exacerbation of asthma fail medical therapy and require urgent intubation and mechanical ventilation. New modalities of ventilatory support, including noninvasive ventilation, have been shown to provide effective ventilation even in the presence of severe bronchoconstriction. An intrinsically high level of auto positive end-expiratory pressure in these patients requires a precise balance between respiratory frequency, tidal volume and inspiratory flow rates. Pressure support ventilation reduces the risk of barotrauma and lowers the work of breathing in these patients. Adjuvant therapy with inhaled anesthetics and bronchoalveolar lavage may also be indicated in patients requiring high pressures to achieve adequate ventilation.

Thoracoabdominal motion in response to treadmill and cycle exercise.

The present study was designed to assess differences in compartmental contributions to ventilation between cycle pedaling and treadmill walking. Eight subjects performed submaximal cycle (CE) and treadmill (TM) exercise. The mean maximal work loads were not significantly different between the 2 modes of exercise. Ventilation (VE), oxygen uptake (VO2), and respiratory inductance plethysmography (RIP) were measured continuously during both modes of exercise. During both TM and CE, VE measured by RIP was found to correlate significantly with VE measured by pneumotachometer. Although the ventilation/work load relationship for CE exceeded that for TM in all subjects, when changes in rib cage (RC) and abdominal (ABD) tidal volume responses were expressed relative to changes in VE, no significant differences were found between TM and CE (RC, p greater than 0.05; ABD, p greater than 0.05). When these changes in tidal volume were expressed relative to changes in work load and independent of ventilation, there were no significant differences between separate compartmental responses during TM or CE (RC, p greater than 0.05; ABD, p greater than 0.05). We conclude that while absolute degrees of ventilation at a given work load differ between cycling and walking, the postural and ventilatory differences between these forms of exercise do not appear to induce differences in the relative contributions of the rib cage or the abdomen to tidal breathing.

Identification and characterization of a high-affinity peripheral-type benzodiazepine receptor in rabbit urinary bladder.

The present study used radioligand binding and in vitro contractility experiments to identify and characterize a peripheral-type benzodiazepine receptor PBR in rabbit urinary bladder. [3H]PK11195 bound to bladder membranes with high-affinity and density (Kd = 5.2 nM., Bmax = 268 fmol./mg. protein), indicating the presence of a PBR. [3H]flunitrazepam bound with high-affinity and density (Kd = 1.2 nM., Bmax = 48 fmol./mg. protein). The rank order potency of various benzodiazepines and isoquinoline carboxamides in displacing the binding of [3H]PK11195 was Ro5-4864 > diazepam = flunitrazepam >> Ro15-1788 = clonazepam. Ro5-4864 and PK11195 inhibited nerve-evoked contractions in a concentration-dependent manner (IC50 = 42 microM. and 56 microM., respectively). Carbachol- and KCl-induced contractions were also inhibited by Ro5-4864 and PK11195. KCl-induced contractions were inhibited to a greater extent than carbachol-induced or field-stimulated contractions with all the drugs tested. Both Ro5-4864 and PK11195 significantly increased the ED50 for calcium-induced contractions following a cholinergic stimulus compared with control. These data demonstrate the presence of a PBR in urinary bladder capable of altering contractility in vitro through modulation of calcium activity.

The effects of transferrin receptor antibody, transferrin receptor antibody bound to Pseudomonas exotoxin and transforming growth factor-alpha bound to Pseudomonas exotoxin on human tenon's capsule fibroblast proliferation.

Pharmacological agents which modulate the wound healing process by the inhibition of proliferation of fibroblasts may improve the success of glaucoma filtration surgery. Since cell proliferation is essential to the wound healing process, we targeted the surface receptors that are associated with proliferating cells. We present the effects of three such agents-purified mouse anti-human transferrin receptor monoclonal antibody 42/6 (anti-TfR-42/6), anti-transferrin monoclonal antibody bound to a Pseudomonas exotoxin (anti-TfR-PE40) and transforming growth factor-alpha Pseudomonas exotoxin (TGF-alpha-PE40)--on human fibroblasts from Tenon's capsule. The inhibition of human subconjunctival fibroblast proliferation by anti-TfR-42/6 (with a concentration up to 25 micrograms/ml) and by anti-TfR-PE40 and TGF-alpha-PE40 (both with a concentration range of 5000-0.00001 micrograms/ml) was determined by colorimetric (OD), and cell counting (CC) assays over a 9-day period. Neither anti-TfR-42/6 nor anti-TfR-PE40 had an antiproliferative effect on the fibroblasts. TGF-alpha-PE40 demonstrated an antiproliferative effect in a dose response manner. The mean 50% inhibitory dose (ID50) by OD was 32.91 micrograms/ml, while the ID50 by CC was 27.88 micrograms/ml. EGF was used as a negative control for TGF-alpha-PE40 toxin. The inhibitory effect of the toxin conjugate was completely blocked by the addition of 1000 micrograms/ml of EGF. These in vitro studies show that TGF-alpha-PE40 may be useful in modulating the proliferation of human ocular fibroblasts; they also give some indication of drug dosages for future in vivo testing.

The effects of 5-fluorouridine, 5-fluorodeoxyuridine, and 5-fluorodeoxyuridine monophosphate on rabbit tenon's capsule fibroblasts in vitro.

Inhibition of rabbit subconjunctival fibroblast attachment and proliferation by 5-fluorouridine (FUR), 5-fluoro-2 deoxyuridine (FUdR), and 5-fluoro-2-deoxyuridine-5-monophosphate (FdUMP), was determined by 3H-adenosine uptake, cell counting, and colorimetric assays for the concentration range of 1000 to 0.0001 micrograms/ml over an 9 day period. The mean 50% inhibitory doses against proliferation were calculated for each assay. Rabbit fibroblast attachment was not inhibited at any drug concentration by either FUR, FUdR, or FdUMP. For rabbit fibroblast proliferation, FUR was found to be 10-100 fold more potent than FUdR and FdUMP. When comparing the human and rabbit cells, the unpaired t-test analysis showed no consistent statistical difference of the ID50s for FUR, FUdR or FdUMP. Rabbit ocular fibroblasts may be useful in modeling the proliferation of human ocular fibroblasts. These in vitro results may be useful for predicting optimal drug dosages for future in vivo testing of these drugs.

The effects of photofrin on human Tenon's capsule fibroblasts in vitro.

Pharmacological agents that modulate the wound healing process by inhibiting fibroblast proliferation may improve the success of proliferative vitreoretinopathy and glaucoma filtration surgery and may have applications in other surgical fields. It is possible that light-absorbing chemicals can be used to cause photo reactions in proliferating fibroblasts as a means of controlling this wound healing process. We present the effects of photofrin porfimer sodium (serial tenfold dilutions 1000-0.00001 micrograms/ml) on human fibroblasts from Tenon's capsule in vitro, with and without photoactivation with Argon green laser (700 mW for 1 and 5 minutes) and with bright sunlight (for 1 and 5 min). The cell density was measured on day 2 by 3H-thymidine uptake and on day 9 by means of a coulter counter, and optical density was measured in terms of the activity of the enzyme hexosaminidase. Each experiment was performed three times in quadruplicate. The counts were averaged for each drug concentration and mean cell count with a standard error as well as the 50% inhibitory doses (ID50s) were calculated. Photofrin demonstrated an inhibitory dose response curve (dark toxicity) to human fibroblasts. Concentrations greater than 100 micrograms/ml of photofrin alone completely inhibited cell growth. Concentrations less than 0.01 micrograms/ml did not have any effect on fibroblast proliferation. There was no significant log dose shift of the inhibitory effect of photofrin with the exposure to either sunlight or Argon laser. Photofrin may be used as a cytotoxic agent alone but does not appear to be activated by light to modulate subconjunctival fibroblast proliferation within the laser parameters used.

Determinants of entry cofactor utilization and tropism in a dualtropic human immunodeficiency virus type 1 primary isolate.

Human immunodeficiency virus type 1 strain 89.6 is a dualtropic isolate that replicates in macrophages and transformed T cells, and its envelope mediates CD4-dependent fusion and entry with CCR5, CXCR-4, and CCR3. To map determinants of cofactor utilization by 89.6 and determine the relationship between cofactor use and tropism, we analyzed recombinants generated between 89.6 and T-cell-tropic (HXB) or macrophage-tropic (JRFL) strains. These chimeras showed that regions of 89.6 env outside V3 through V5 determine CXCR-4 utilization and T-cell line tropism as well as CCR5 utilization and macrophage tropism. However, the 89.6 env V3 domain also conferred on HXB the ability to use CCR5 for fusion and entry but not the ability to establish productive macrophage infection. CCR3 use was conferred on HXB by 89.6 env V3 or V3 through V5 sequences. While replacement of the 89.6 V3 through V5 region with HXB sequences abrogated CCR3 utilization, replacement of V3 or V4 through V5 separately did not. Thus, CCR3 use is determined by sequences within V3 through V5 and most likely can be conferred by either the V3 or the V4 through V5 domains. These results indicate that cofactor utilization and tropism in this dualtropic isolate are determined by complex interactions among multiple env segments, that distinct regions of the Env glycoprotein may be important for utilization of different chemokine receptors, and that determinants in addition to cofactor usage participate in postentry stages in the virus replication cycle that contribute to target cell tropism.

Pulmonary function in adolescents with mild idiopathic scoliosis.

Spirometric indices, lung volumes, maximum voluntary ventilation, and maximum inspiratory and expiratory pressures were measured in 44 adolescents with mild idiopathic scoliosis (spinal curvature less than 30 degrees). All were symptom free, but six (13.6%) showed a restrictive defect with forced vital capacity less than 80% of predicted. In 12 subjects (27.3%) maximum voluntary ventilation was reduced to less than 80% of predicted normal. Forced vital capacity was significantly correlated with maximum inspiratory pressure and with maximum expiratory pressure, measures of respiratory muscle strength, but was not related to the degree of thoracic curvature. When maximum inspiratory pressure and forced vital capacity were corrected for differences in body size these variables remained positively correlated, most significantly in the girls. These data indicate that ventilatory function may be impaired in mild, idiopathic scoliosis and that the force developed by the respiratory muscles is a more important determinant of this impairment than the radiologically determined degree of spinal curvature.

Human prostate muscarinic receptor subtypes.

The alpha adrenergic receptor subtypes of the human prostate have been intensively investigated, while the muscarinic receptor subtypes and their function have yet to be determined in this tissue. [3H]-QNB binding to muscarinic receptors was performed on membrane homogenates of adenoma from six prostatectomy specimens resulting in an average total receptor density of 46 fMol/mg protein. Pirenzepine, hexahydrosiladifenidol, and para-fluoro-hexahydrosiladifenidol, drugs with high affinity for the M1 subtype, were significantly more potent inhibitors of [3H]-QNB binding than the M2 selective drug methoctramine. Immunoprecipitation studies were done using antisera raised to individual M1-M5 receptor subtypes. Approximately 75% of the solubilized receptors in the adenoma specimens were immunoprecipitated with the anti-M1 antibody, in contrast to 5% or less with antibodies against M2, M3 or M4 subtypes. These immunoprecipitation studies confirm the preponderance of the M1 subtype in prostate adenoma suggested by the high affinity pirenzepine binding. M1 receptors, when incubated with agonist, coimmunoprecipitated with the alpha subunits of the guanine nucleotide binding regulatory proteins Gi alpha, Gq/11 alpha and G16 alpha. Immunohistochemical staining with the anti-M1 antibody demonstrates the M1 receptor to be localized to the glandular epithelium. The human prostate is the first peripheral tissue in which a preponderance of the M1 subtype of muscarinic receptors has been demonstrated.

A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors.

Here, we show that the beta-chemokine receptor CKR-5 serves as a cofactor for M-tropic HIV viruses. Expression of CKR-5 with CD4 enables nonpermissive cells to form syncytia with cells expressing M-tropic, but not T-tropic, HIV-1 env proteins. Expression of CKR-5 and CD4 enables entry of a M-tropic, but not a T-tropic, virus strain. A dual-tropic primary HIV-1 isolate (89.6) utilizes both Fusin and CKR-5 as entry cofactors. Cells expressing the 89.6 env protein form syncytia with QT6 cells expressing CD4 and either Fusin or CKR-5. The beta-chemokine receptors CKR-3 and CKR-2b support HIV-1 89.6 env-mediated syncytia formation but do not support fusion by any of the T-tropic or M-tropic strains tested. Our results suggest that the T-tropic viruses characteristic of disease progression may evolve from purely M-tropic viruses prevalent early in virus infection through changes in the env protein that enable the virus to use multiple entry cofactors.

Regions in beta-chemokine receptors CCR5 and CCR2b that determine HIV-1 cofactor specificity.

Macrophage-tropic (M-tropic) HIV-1 strains use the beta-chemokine receptor CCR5, but not CCR2b, as a cofactor for membrane fusion and infection, while the dual-tropic strain 89.6 uses both. CCR5/2b chimeras and mutants were used to map regions of CCR5 important for cofactor function and specificity. M-tropic strains required either the amino-terminal domain or the first extracellular loop of CCR5. A CCR2b chimera containing the first 20 N-terminal residues of CCR5 supported M-tropic envelope protein fusion. Amino-terminal truncations of CCR5/CCR2b chimeras indicated that residues 2-5 are important for M-tropic viruses, while 89.6 is dependent on residues 6-9. The identification of multiple functionally important regions in CCR5, coupled with differences in how CCR5 is used by M- and dual-tropic viruses, suggests that interactions between HIV-1 and entry cofactors are conformationally complex.