Elliot M. Landaw

Transport Across the Primate Blood-Brain Barrier of a Genetically Engineered Chimeric Monoclonal Antibody to the Human Insulin Receptor

AbstractPurpose. Brain drug targeting may be achieved by conjugating drugs,that normally do not cross the blood-brain barrier (BBB), to brain drugdelivery vectors. The murine 83-14 MAb to the human insulin receptor(HIR) is a potential brain drug targeting vector that could be used inhumans, if this MAb was genetically engineered to form a chimericantibody, where most of the immunogenic murine sequences arereplaced by human antibody sequence. Methods. The present studies describe the production of the gene forthe chimeric HIRMAb, expression and characterization of the protein,radiolabeling of the chimeric HIRMAb with 111-indium and125-iodine, and quantitative autoradiography of living primate brain taken 2hours after intravenous administration of the [111In]chimeric HIRMAb. Results. The chimeric HIRMAb had identical affinity to the targetantigen as the murine HIRMAb based on Western blotting andimmunoradiometric assay using partially purified HIR affinity purified fromserum free conditioned media produced by a CHO cell line secretingsoluble HIR. The [125I]chimeric HIRMAb was avidly bound to isolatedhuman brain capillaries, and this binding was blocked by the murineHIRMAb. The [111In]chimeric HIRMAb was administeredintravenously to an anesthetized Rhesus monkey, and the 2 hour brain scanshowed robust uptake of the chimeric antibody by the living primatebrain. Conclusions. A genetically engineered chimeric HIRMAb has beenproduced, and the chimeric antibody has identical reactivity to thehuman and primate BBB HIR as the original murine antibody. Thischimeric HIRMAb may be used in humans for drug targeting throughthe BBB of neurodiagnostic or neurotherapeutic drugs that normallydo not cross the BBB.

Use of Electrochemical DNA Biosensors for Rapid Molecular Identification of Uropathogens in Clinical Urine Specimens

ABSTRACT We describe the first species-specific detection of bacterial pathogens in human clinical fluid samples using a microfabricated electrochemical sensor array. Each of the 16 sensors in the array consisted of three single-layer gold electrodes—working, reference, and auxiliary. Each of the working electrodes contained one representative from a library of capture probes, each specific for a clinically relevant bacterial urinary pathogen. The library included probes for Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterocococcus spp., and the Klebsiella-Enterobacter group. A bacterial 16S rRNA target derived from single-step bacterial lysis was hybridized both to the biotin-modified capture probe on the sensor surface and to a second, fluorescein-modified detector probe. Detection of the target-probe hybrids was achieved through binding of a horseradish peroxidase (HRP)-conjugated anti-fluorescein antibody to the detector probe. Amperometric measurement of the catalyzed HRP reaction was obtained at a fixed potential of −200 mV between the working and reference electrodes. Species-specific detection of as few as 2,600 uropathogenic bacteria in culture, inoculated urine, and clinical urine samples was achieved within 45 min from the beginning of sample processing. In a feasibility study of this amperometric detection system using blinded clinical urine specimens, the sensor array had 100% sensitivity for direct detection of gram-negative bacteria without nucleic acid purification or amplification. Identification was demonstrated for 98% of gram-negative bacteria for which species-specific probes were available. When combined with a microfluidics-based sample preparation module, the integrated system could serve as a point-of-care device for rapid diagnosis of urinary tract infections.

Persistence of insulin resistance in polycystic ovarian disease after inhibition of ovarian steroid secretion.

Six nonobese women with polycystic ovarian disease (PCOD) showed significant hyperinsulinemia, compared with controls after oral glucose (P less than 0.05). As an indicator of insulin sensitivity, in vitro proliferation of erythrocyte progenitor cells of PCOD subjects exposed to physiologic concentrations of insulin was significantly blunted (P less than 0.001). Monocyte insulin receptor binding was not impaired in the PCOD subjects. Three of the PCOD patients were treated with a long-acting gonadotropin-releasing hormone agonist for 6 months, which resulted in marked suppression of ovarian androgen secretion but no demonstrable changes in in vivo or in vitro indicators of insulin resistance. Thus insulin resistance in PCOD subjects appears to be unrelated to ovarian hyperandrogenism (or acanthosis or obesity). Although certain tissues are insulin-resistant in PCOD patients, the ovary may remain sensitive and overproduce androgens in response to high circulating insulin levels.

Phosphorylation of the ATP-binding loop directs oncogenicity of drug-resistant BCR-ABL mutants

The success of targeting kinases in cancer with small molecule inhibitors has been tempered by the emergence of drug-resistant kinase domain mutations. In patients with chronic myeloid leukemia treated with ABL inhibitors, BCR-ABL kinase domain mutations are the principal mechanism of relapse. Certain mutations are occasionally detected before treatment, suggesting increased fitness relative to wild-type p210 BCR-ABL. We evaluated the oncogenicity of eight kinase inhibitor-resistant BCR-ABL mutants and found a spectrum of potencies greater or less than p210. Although most fitness alterations correlate with changes in kinase activity, this is not the case with the T315I BCR-ABL mutation that confers clinical resistance to all currently approved ABL kinase inhibitors. Through global phosphoproteome analysis, we identified a unique phosphosubstrate signature associated with each drug-resistant allele, including a shift in phosphorylation of two tyrosines (Tyr253 and Tyr257) in the ATP binding loop (P-loop) of BCR-ABL when Thr315 is Ile or Ala. Mutational analysis of these tyrosines in the context of Thr315 mutations demonstrates that the identity of the gatekeeper residue impacts oncogenicity by altered P-loop phosphorylation. Therefore, mutations that confer clinical resistance to kinase inhibitors can substantially alter kinase function and confer novel biological properties that may impact disease progression.

Tracer kinetic model of blood-brain barrier transport of plasma protein-bound ligands. Empiric testing of the free hormone hypothesis.

Previous studies have shown that the fraction of hormone or drug that is plasma protein bound is readily available for transport through the brain endothelial wall, i.e., the blood-brain barrier (BBB). To test whether these observations are reconcilable with the free-hormone hypothesis, a tracer-kinetic model is used in the present investigations to analyze in vivo initial extraction data on BBB transport of protein-bound steroid hormones (dihydrotestosterone, testosterone, estradiol, and corticosterone), thyroid hormones (triiodothyronine), and lipophilic amine drugs (propranolol). The plasma proteins used are bovine albumin and human orosomucoid. Transport data was fit to a modification of the Kety-Renkin-Crone equation of capillary physiology; the modified equation incorporates the principles of both capillary physiology and plasma protein-ligand mass action binding relationships. In most cases, the experimental data is best fit to the model equation when the apparent in vivo dissociation constant, KDa, of the ligand protein binding reaction increases to values that are 5- to 50-fold greater than the in vitro dissociation constant, KD. This result indicates that the rate of ligand dissociation from the plasma protein is accelerated in the capillary bed relative to the in vitro situation. It is hypothesized that the major factor leading to the rapid transport in vivo of protein-bound ligands into tissues such as brain is an endothelial-induced decrease in the affinity of the plasma protein for the ligand. Under these conditions, the amount of plasma ligand available for tissue clearance in vivo parallels the protein-bound fraction, not the free hormone.

Measuring the genetic contribution of a single locus to a multilocus disease

An increasing number of diseases are being demonstrated to be due to determinants at more than one genetic locus. It thus becomes of interest to determine the genetic contribution of a specific single locus. A method of estimating a “coefficient of genetic contribution” is described herein, based on a comparison of monozygotic twin concordance data for a specific disease, the empirical sibling recurrence risks, and the sharing of identical by descent genes at the specific locus of interest by pairs of siblings who are both affected. The value of the method is that it requires relatively few assumptions, and does not require knowledge of the mode of inheritance of disease susceptibility at the gene locus of interest. If there are major environmental determinants, this method will give a lower bound for the single locus of interest. To illustrate the method, it is applied to two specific diseases, insulin dependent diabetes mellitus (IDDM) and gluten‐sensitive enteropathy (GSE), and a specific locus, the HLA gene complex. The best estimates would appear to be that the HLA “genes” provide a coefficient of 60% for IDDM susceptibility, but only 30% for GSE. A possible reason for these differences is the markedly increased disease susceptibility of the DR3/DR4 heterozygote for IDDM.

Selecting donors of platelets for refractory patientson the basis of HLA antibody specificity

BACKGROUND: Patients who are refractory to platelet transfusion as a result of HLA alloimmunization are generally given HLA‐matched or crossmatched platelets. However, HLA‐matched platelets that are matched at HLA‐A and ‐B loci (A‐matched) or those without any mismatched or cross‐reactive antigens (BU‐matched) are frequently unavailable. A disadvantage of crossmatching is that crossmatched platelets have a shelf life of only 5 days, so that crossmatch tests must be performed frequently for patients requiring long‐term platelet transfusions. An alternative method is the selection of platelets according to the patients HLA antibody specificity, called the antibody specificity prediction (ASP) method.

Carotid Artery Injection Technique: Bounds for Bolus Mixing by Plasma and by Brain

Estimation of Michaelis-Menten kinetic parameters (Km, Vmax) of blood–brain barrier (BBB) transport processes with the carotid artery single injection technique assumes that mixing of the bolus with unlabeled substrate either from (a) circulating plasma or (b) amino acid efflux from brain, is minimal. The maximum extent to which the bolus could mix by these two sources is quantified in the present studies by measuring 14C-phenylalanine extraction in pentobarbital-anesthetized and conscious rats after the addition of 0–80% rat serum to the arterial injection solution. An upper bound (±SE) of bolus mixing due to mixing from both sources, expressed in terms of percentage of rat plasma, is 8.8 ± 1.9 and 7.0 ± 2.1% for the anesthetized and conscious rat, respectively. The estimated contribution to bolus mixing due to amino acid efflux from brain is 3.3 and 2.1% for the anesthetized and conscious rat, respectively. Based on these estimates, the upper bound for bolus mixing with circulating rat plasma is only 5.5 and 4.9%, respectively, for the anesthetized and conscious catheterized rat. Thus, any bolus mixing after rapid carotid injection is relatively small and is comparable to the mixing effects observed with the carotid artery infusion technique. Mixing effects on the order of 5% are shown to have no significant effect on the estimation of kinetic parameters of BBB nutrient transport, except for neutral and basic amino acid transport, which are characterized by very low Km values relative to the usual amino acid plasma concentrations. In the rat, a 5% mixing results in an enrichment of the bolus concentration of unlabeled amino acid that approximates the Km of the transport process, and this results in an overestimation of the absolute Km value. However, mixing effects are shown to have little, if any, impact on the estimation of the transport Vmax, KD, or apparent Km. Thus, amino acid influx rates predicted from kinetic constants obtained with the carotid injection technique are reliable, even if bolus mixing effects with the carotid injection technique are as high as 7–9%.

Interictal Seizure Resections Show Two Configurations of Endothelial Glut1 Glucose Transporter in the Human Blood–Brain Barrier

Immunogold electron microscopy was used to analyze and quantify the Glut1 glucose transporter in brain tissue from five patients undergoing surgery for treatment of seizures. Samples were prepared from two different regions of each resection: (1) the most actively spiking epileptogenic site, and (2) the least actively spiking region, as indicated by intraoperative EEG monitoring. Two configurations of endothelial cell Glut1 were observed. About one half of the capillary profiles examined displayed abundant Glut1 immunoreactivity on both luminal and abluminal endothelial membranes. In the remainder of the profiles, reduced Glut1 labeling was seen, but adjacent erythrocyte membranes remained highly Glut1 immunoreactive, suggesting that reduced endothelial Glut1 reactivity was not attributable to method artifacts. Immunogold studies using antisera to human glial fibrillary acidic protein and human serum albumin demonstrated increased quantities of these two epitopes in the extravascular regions in which more EEG spiking activity had been demonstrated. These observations were consistent with the hypotheses that capillary integrity was more compromised, and gliosis was quantitatively increased, in the more actively spiking region of the resection. Altered glucose transporter activity in the blood–brain barrier was characterized by a bimodal Glut1 distribution in which the smaller (type B) endothelial cells displayed low Glut1 immunoreactivity, whereas adjacent (and even contiguous) larger (type A) endothelial cells showed 5- to 10-fold greater expression of membrane Glut1 transporter protein. Because this transporter facilitates glucose entry to the brain, small pericapillary volumes of brain tissue may have quite different concentrations of glucose. We hypothesize that in complex partial seizures and other forms of brain insult, an alteration of blood–brain barrier Glut1 glucose transporter activity is indicated by the appearance of these two subpopulations of endothelial cells. In comparison with previous studies of human brain capillaries in hemangioblastoma and brain injury, endothelial Glut1 density was apparently reduced (interictally) in affected temporal lobes of patients with complex partial seizures.

survival outcome among 54 intubated pediatric bone marrow transplant patients

Objectives: To assess the outcome of children who required endotracheal intubation after bone marrow transplantation and to determine whether prognostic indicators that might assist decision‐making regarding the institution of mechanical ventilation could be identified. Design: Retrospective chart review. Setting: Critical care, reverse isolation unit at a university hospital. Patients: Fifty‐four pediatric bone marrow transplant recipients who required endotracheal intubation. Interventions: None. Measurements and Main Results: The following variables were assessed for effect on survival: a) the presence of additional nonhematoporetic organ system failure; b) the duration of required ventilatory assistance; c) the etiology of respiratory failure; d) the presence of significant graft vs. host disease; and e) the underlying disease for which the transplant was done. Six of 54 intubated pediatric bone marrow transplant recipients were extubated and discharged from the hospital. No patient with a diagnosis of leukemia or with multiple organ system failure could be extubated or discharged from the hospital. The presence of pulmonary parenchymal disease indicated poor prognosis for survival. Conclusions: The decision to intubate a pediatric bone marrow transplant patient remains a difficult one. In this population, multiple organ system failure and primary pulmonary parenchymal disease were associated with a high mortality rate. These factors should be taken into account before and throughout the course of mechanical ventilation in this patient population. (Crit Care Med 1994; 22:171‐176)