Treating cat allergy with monoclonal IgG antibodies that bind allergen and prevent IgE engagement.

Acute allergic symptoms are caused by allergen-induced crosslinking of allergen-specific immunoglobulin E (IgE) bound to Fc-epsilon receptors on effector cells. Desensitization with allergen-specific immunotherapy (SIT) has been used for over a century, but the dominant protective mechanism remains unclear. One consistent observation is increased allergen-specific IgG, thought to competitively block allergen binding to IgE. Here we show that the blocking potency of the IgG response to Cat-SIT is heterogeneous. Next, using two potent, pre-selected allergen-blocking monoclonal IgG antibodies against the immunodominant cat allergen Fel d 1, we demonstrate that increasing the IgG/IgE ratio reduces the allergic response in mice and in cat-allergic patients: a single dose of blocking IgG reduces clinical symptoms in response to nasal provocation (ANCOVA, p = 0.0003), with a magnitude observed at day 8 similar to that reported with years of conventional SIT. This study suggests that simply augmenting the blocking IgG/IgE ratio may reverse allergy.

Point mutations in the beta chain CDR3 can alter the T cell receptor recognition pattern on an MHC class I/peptide complex over a broad interface area.

To study how the T cell receptor interacts with its cognate ligand, the MHC/peptide complex, we used site directed mutagenesis to generate single point mutants that alter amino acids in the CDR3beta loop of a H-2Kb restricted TCR (N30.7) specific for an immunodominant peptide N52-N59 (VSV8) derived from the vesicular stomatitis virus nucleocapsid. The effect of each mutation on antigen recognition was analyzed using wild type H-2Kb and VSV8 peptide, as well as H-2Kb and VSV8 variants carrying single replacements at residues known to be exposed to the TCR. These analyses revealed that point mutations at some positions in the CDR3beta loop abrogated recognition entirely, while mutations at other CDR3beta positions caused an altered pattern of antigen recognition over a broad area on the MHC/peptide surface. This area included the N-terminus of the peptide, as well as residues of the MHC alpha1 and alpha2 helices flanking this region. Assuming that the N30 TCR docks on the MHC/peptide with an orientation similar to that recently observed in two different TCR-MHC/peptide crystal structures, our findings would suggest that single amino acid alterations within CDR3beta can affect the interaction of the TCR with an MHC surface region distal from the predicted CDR3beta-Kb/VSV8 interface. Such unique recognition capabilities are generated with minimal alterations in the CDR3 loops of the TCR. These observations suggest the hypothesis that extensive changes in the recognition pattern due to small perturbations in the CDR3 structure appears to be a structural strategy for generating a highly diversified TCR repertoire with specificity for a wide variety of antigens.

Some observations on the course and relations of the iliohypogastric and ilioinguinal nerves (based on 348 specimens).

In a series of 174 dissecting-room embalmed human cadavers, of both sexes, observations have been made on the variations in the course and relations of the iliohypogastric and ilioinguinal nerves. The mean distance of both the nerves and ot their common trunk (whenever present) from the middle of the inguinal ligament has been found to be as follows:: Iliohypogastric nerve; 2.70 cm (2.75 cm on the right side and 2.66 cm on the left side), ilioinguinal nerve; 0.64 cm (0.62 cm on the right side and 0.67 cm on the left side), and common trunk 0.62 cm (0.70 cm on the right and 0.54 cm on the left side). The position of the iliohypogastric nerve was less than 2.0 cm from the middle of the inguinal ligament in 22.21% of the cases. Conversely, the position of the ilioinguinal nerve was greater than 1 cm from the middle of the inguinal ligament in 23% of the cases. The distance between the emergence of these nerves subcutaneously and the linea alba is not constant. The data obtained from the measurements were not statistically different with regard to either side or sex. Nevertheless, the knowledge of the variations in the course of the iliohypogastric and ilioinguinal nerves and of their common trunk is of practical clinical importance.

Anatomical observations on the intermediate ganglion of the cervical sympathetic trunk (based on 232 specimens).

In a series of 116 adult dissecting-room human cadavers, the frequency, location and branches of the intermediate ganglion of the cervical sympathetic trunk were studied, bilaterally, i.e. in 232 specimens. The frequency of the ganglion was found 24.14%, of which in 12.93% a middle cervical ganglion was also present. The intermediate ganglion, lying usually anterior to the transverse process of the seventh cervical vertebra (41.2%) or to that of the sixth (35.3%), gives off splachnic and vascular branches and rami communicantes to the 6th and 7th cervical nerves. Its rami internodiales contribute to the prevertebral and subclavian ansae. Because of its independent preganglionic fibers the knowledge of its frequency and surgical anatomy is of significance in gangliectomies.

The relations of the superior pole of the stellate ganglion to the vertebral artery.

The relations of the superior pole of the stellate ganglion to the vertebral artery and to the transverse processes of the last cervical and the first thoracic vertebrae were studied in 440 specimens (220 adult human embalmed cadavers). The superior pole of the ganglion was found, most commonly, within an "arterial triangle" bordered by the subclavian, the common carotid and the vertebral arteries (75.8%), at the level of the inferior third of the vertebral artery (79.8%) and anterior to the interval between the transverse process of the seventh cervical and the first thoracic vertebrae (48.6%). The variations of these locations of the stellate ganglion, found in this study, may be of significant surgical anatomical importance.

Effect of nonionizing radiation on the Purkinje cells of the uvula in squirrel monkey cerebellum.

Pregnant squirrel monkeys were exposed to 2450-MHz (CW) microwaves at an equivalent power density of 10 mW/cm2 (SAR 3.4 mW/g) for three hours daily in a cavity-cage module. The exposure began when pregnancy was determined by a hormonal method, and continued through of offspring's first 9.5 months. After irradiation, the brain of the offspring were fixed with formaldehyde, and the inferior vermis of each cerebella was removed and processed for histologic observations. Purkinje cell density in the uvula was determined in sagittal serial section. There was no significant difference between control and experimental animals in the number of Purkinje cells per mm of Purkinje cell line (linear density), as well as in the density of Purkinje cells in the Purkinje cell layer.

Some quantitative histological observations on the growing cerebellum of the albino rat. (Dimensions of the cortical layers and white core and relative number of Purkinje cells).

The cerebella of 38 Sprague Dawley rats of various ages have been studied histologically by sectioning in certain parasagittal planes. The age groups were chosen from the early postnatal developmental stage (6, 11 and 16 days), around puberty (41, 46 and 51 days) and maturity (210 days). The sections yielded quantitative data with regard to the dimensions in parasagittal sections, of the entire cerebellum, the cerebellar cortex, the white core, the molecular layer and the granular layer, as well as, the differential and proportional growth of these layers at the above ages. The relative number of Purkinje cells per mm2 of sectional area of the cortex was also determined. The information presented in this study is of indicative value of the pattern of the growing cerebellum and of the ages in which the various layers and the relative number of Purkinje cells assume adult values.

Fiber contribution from the mesencephalic nucleus of the trigeminal nerve to the trochlear nerve in the cat: a histological quantitative study.

The fiber connections between the trigeminal mesencephalic nucleus and tract and the trochlear nerve root of 15 cats were examined after silver impregnation of the pontomescencephalic region of the brains. The results revealed that: (a) some of the mesencephalic root fascicles join the trochlear root, (b) some of the mesencephalic root cells contribute their processes to the trochlear root, and (c) some mesencephalic cells are found amidst the fibers of the trochlear nerve during its intrabulbar course. The fibers of the trochlear nerve were counted at certain preselected sites before and after crossing the mesencephalic nucleus. The statistical data obtained indicated that the trigeminal mesencephalic root contributes 4-10% of the fibers of the trochlear nerve, before it crosses the mesencephalic nucleus.

A fascial canal for the great saphenous vein: gross and microanatomical observations.

The course of the great saphenous vein in a fascial canal derived from the deep fascia of the lower extremity is described. The fascial canal is absent in 15% of the cases. In 85% of the cases, the sheath surrounding the great saphenous vein is continuous from the thigh to the leg and supports the vein from the hydrostatic pressure of the blood, being absent only in the uppermost and the lowermost quarters of the vein in the thigh and the leg, respectively. Thus, up to one quarter of the vein is ensheathed in 30%, up to one half in 45% and up to three quarters in 10% of the cases studied. The absence of this fascial envelope of the vein, in whole or in part, may be involved in the pathogenesis or the complications of the varicosities of the great saphenous vein along with the well-known aggravating factors.

Electron spin-echo studies of the copper(II) binding sites in dopamine beta-hydroxylase.

The active site structure of Cu(II) in dopamine beta-hydroxylase, isolated from bovine adrenal medulla, was studied by pulsed electron paramagnetic resonance (EPR) spectroscopy. Fourier transformation of the stimulated electron spin-echo envelope revealed frequency components characteristic of Cu(II)-histidyl imidazole coordination. The three major lines in the spectrum at 0.7, 1.4, and 4.0 MHz are typical for Cu(II)-imidazole complexes where imidazole is protonated and equatorially coordinated. Quantitation of the number of imidazole ligands bound to Cu(II) in enzyme containing two, four, and eight Cu per protein tetramer, as well as characterization of the superhyperfine coupling parameters, was achieved by spectral simulation. In all cases, it was shown that there are three, or more likely four, imidazole ligands bound to Cu(II). Addition of deuteriated substrate analogues to the enzyme did not produce any observable deuterium modulation in the spin-echo envelopes, thus indicating that the distance between substrate deuterons and Cu(II) is greater than 5 A.

Characterization of highly purified dopamine beta-hydroxylase.

A modified purification procedure has been developed for dopamine beta-hydroxylase isolated from bovine adrenal medulla. Catalase is included in the homogenization step starting with a suspension of either chromaffin granules or adrenal medulla tissue. With this precaution, the enzyme remains stable in the supernatant solution in preparation for the subsequent purification step involving concanavalin A-Sepharose chromatography. The homogeneous enzyme has a specific activity in the range of 60-70 mumol O2 consumed/min/mg. Using radiolabeled metal ion chelators, it was determined that several of the chelators remained tightly bound to the enzyme after removal of the copper leading to difficulties in establishing stoichiometry of enzyme-bound metal ions.

A quantitative estimation of the divergence between the trabecular system and the stress trajectories in the upper end of the human femoral bone.

In the frame of the widely accepted theory that the internal architecture of the cancellous part of the bones is strongly influenced by the mechanical stresses that predominate inside them, a quantification of the convergence between the direction of the principal stresses and that of the trabeculae has been attempted, by measuring the angle between these 2 directions, in frontal sections of the upper end of a series of femoral bones (left and right), taken out from embalmed human cadavers. This study suggests that there is a fairly good convergence between trabeculae and stress (compressive as well as tensile), since the angles which have been measured have a mean value of +/- 7 degrees. It is further observed that this convergence is better (i.e. the angle is lesser) in those parts of the section where the corresponding stress (compressive or tensile) is greater. This result may prove particularly useful in connection to the effort of elucidating the mechanism that governs the influence of bone stress to osteogenesis.

Kinetic and spectroscopic studies of the interaction of copper with dopamine beta-hydroxylase.

The question of the stoichiometry of copper bound to dopamine beta-hydroxylase and the number of copper atoms required for maximal activity was addressed in this study. Incubation of tetrameric enzyme from bovine adrenal medulla with 64Cu2+ followed by rapid gel filtration yielded an enzyme containing 8.3-8.9 mol of Cu/mol of tetramer. An identical stoichiometry was obtained by analysis of bound copper by atomic absorption methods. NMR and EPR were used to monitor titrations of the enzyme with Cu2+ and showed that the longitudinal relaxation rate of solvent water protons and the amplitude of the signal at g approximately 2 increased linearly up to a copper to protein ratio of approximately 8. Additional titrations also indicate that an enzyme-Cu2+-tyramine-CN- inhibitory complex was formed when 8 mol of Cu2+ are bound per mol of enzyme. The rate of inactivation of dopamine beta-hydroxylase by the mechanism-based inhibitor 2-Br-3-(p-hydroxyphenyl)-1-propene was measured and used as a method to follow enzymatic catalysis. An increase in rate was observed with increasing Cu2+ up to a protein to Cu2+ ratio of 8 Cu/tetramer. The rate becomes constant after this ratio is achieved. These data indicate that dopamine beta-hydroxylase specifically binds 8 mol of Cu/tetramer and that this stoichiometry is required for maximal activity.

Binding of longer peptides to the H-2Kb heterodimer is restricted to peptides extended at their C terminus: refinement of the inherent MHC class I peptide binding criteria.

MHC class I molecules usually bind short peptides of 8-10 amino acids, and binding is dependent on allele-specific anchor residues. However, in a number of cellular systems, class I molecules have been found containing peptides longer than the canonical size. To understand the structural requirements for MHC binding of longer peptides, we used an in vitro class I MHC folding assay to examine peptide variants of the antigenic VSV 8 mer core peptide containing length extensions at either their N or C terminus. This approach allowed us to determine the ability of each peptide to productively form Kb/beta2-microglobulin/peptide complexes. We found that H-2Kb molecules can accommodate extended peptides, but only if the extension occurs at the C-terminal peptide end, and that hydrophobic flanking regions are preferred. Peptides extended at their N terminus did not promote productive formation of the trimolecular complex. A structural basis for such findings comes from molecular modeling of a H-2Kb/12 mer complex and comparative analysis of MHC class I structures. These analyses revealed that structural constraints in the A pocket of the class I peptide binding groove hinder the binding of N-terminal-extended peptides, whereas structural features at the C-terminal peptide residue pocket allow C-terminal peptide extensions to reach out of the cleft. These findings broaden our understanding of the inherent peptide binding and epitope selection criteria of the MHC class I molecule. Core peptides extended at their N terminus cannot bind, but peptide extensions at the C terminus are tolerated.

Effect of nonionizing radiation on the Purkinje cells of the rat cerebellum.

In one experiment, Sprague Dawley rats (16-21 days of gestation) and their offspring were exposed to 100-MHz (CW) electromagnetic radiation at 46 mW/cm2 (SAR 2.77 mW/g) for 4 h/day for 97 days. In another experiment, the pregnant rats were irradiated daily from 17 to 21 days of gestation with 2450-MHz (CW) microwaves at 10 mW/cm2 (SAR 2 m W/G) for 21 h/day. In a third experiment, 6-day old rat pups were irradiated 7 h/day for five days with 2450-MHz radiation at 10 mW/cm2. Equal numbers of animals were sham irradiated in each group. Quantitative studies of Purkinje cells showed a significant and irreversible decrease in rats irradiated during fetal or fetal and early postnatal life. In animals exposed postnatally, and euthanized immediately after irradiation, significant decrease in the relative number of Purkinje cells was apparent. However, restoration apparently occurred after forty days of recovery.

An in vitro study of the dynamic features of the major histocompatibility complex class I complex relevant to its role as a versatile peptide-receptive molecule.

The major histocompatibility complex class I complex consists of a heavy chain and a light chain (beta2-microglobulin, beta2m), which assemble with a short endogenously derived peptide in the endoplasmic reticulum. The class I peptide can be directly exchanged, either at the cell surface or, as recently described, in vesicles of the endocytic compartments, thus allowing exogenous peptides to enter the class I presentation pathway. To probe the interactions between the components of the class I molecule, we analyzed the exchange of peptide and beta2m by using purified, recombinant H2-Kb/peptide complexes in a cell-free in vitro system. The exchange of competitor peptide was primarily dependent on the off-rate of the original peptide in the class I binding groove. Peptide exchange was not enhanced by the presence of exogenous beta2m, as exchange occurred to the same extent in its absence. Thus, the exchange of peptide and beta2m are independent events. The exchange rate of beta2m also was not affected by the dissociation rates of the original peptides. Furthermore, peptides could substantially exchange into class I molecules over a pH range of 5.5 to 7.5, conditions prevalent in certain endocytic compartments. We conclude that the dynamic properties of the components of class I molecules explain its function as a highly peptide-receptive molecule. The major histocompatibility complex class I can readily receive peptides independent of the presence of exogenous beta2m, even at a low pH. Such properties are relevant to class I peptide acquisition, which can occur at the cell surface, as well as in specialized endosomes.

Primary sarcomas of the kidney. A clinicopathologic and DNA flow cytometric study of 17 cases.

Primary sarcomas of the kidney in adults are rare. In the handful of published reports of all soft tissue sarcomas, DNA ploidy has correlated with histologic grade and outcome. This report presents the clinicopathologic and flow cytometric features of 17 cases of primary renal sarcoma (seven men and ten women, ages 28-69 years). Presenting symptoms included abdominal and back pain and hematuria. Stages at diagnosis were I, in three patients; II, five patients; III, two patients; and IV, two patients. Eight tumors were leiomyosarcoma, two malignant fibrous histiocytoma, one hemangiopericytoma, one fibrosarcoma, and five unclassified. Tumors measured 5.5 to 23 cm, seven contained marked nuclear pleomorphism, seven were extensively necrotic, and mitotic rate was 1 to 33 per 10 high-power fields. Seven tumors showed aneuploidy and five were diploid. Thirteen patients were dead of disease after a mean of 23 months and two were alive with known metastases at 29 and 33 months, respectively. Ploidy pattern and outcome or time to death were not correlated, but aneuploidy correlated with histologic grade, marked nuclear pleomorphism (P less than 0.05), extensive necrosis (p less than 0.01), and high mitotic rate (0.05 less than P less than 0.10). The authors conclude that although DNA ploidy does correlate with histologic grade, for primary renal sarcomas, whose prognosis in this series was extremely poor, it does not correlate with outcome.

Adult Wilms' tumor: a clinicopathologic study of 11 cases.

Wilms' tumor, one of the most common solid tumors in children, may also arise in the kidney of adults. We report here eleven cases seen in patients greater than 18 yr of age at The University of Texas M. D. Anderson Cancer Center, Houston. There were eight males and three females with ages ranging from 21 to 67 yr with a mean of 32 yr. Gross hematuria and/or an abdominal mass were the most common clinical presentations. The patients presented at a later stage than is typical of children (45% with stages III and IV disease), and eight of ten patients (80%) with adequate follow-up died of disease between 12 and 69 mo (mean, 30 mo; median, 25 mo), despite combined treatment with surgery, chemotherapy, and/or irradiation. Pathologically, a classic triphasic pattern was seen in seven cases and a biphasic pattern in four. None of the tumors showed features of anaplasia. Large tumor size and a high mitotic rate were associated with a poor prognosis. In contrast to cases in children, persistent renal blastema was not found in any of our cases, suggesting that the purported histogenesis of these tumors in childhood may not hold true in adults.