Efficacy of a therapeutic cocaine vaccine in rodent models.

Cocaine abuse is a major medical and public health concern in the United States, with approximately 2.1 million people dependent on cocaine. Pharmacological approaches to the treatment of cocaine addiction have thus far been disappointing, and new therapies are urgently needed. This paper describes an immunological approach to cocaine addiction. Antibody therapy for neutralization of abused drugs has been described previously, including a recent paper demonstrating the induction of anti-cocaine antibodies. However, both the rapidity of entry of cocaine into the brain and the high doses of cocaine frequently encountered have created challenges for an antibody-based therapy. Here we demonstrate that antibodies are efficacious in an animal model of addiction. Intravenous cocaine self-administration in rats was inhibited by passive transfer of an anti-cocaine monoclonal antibody. To actively induce anti-cocaine antibodies, a cocaine vaccine was developed that generated a high-titer, long-lasting antibody response in mice. Immunized mice displayed a significant change in cocaine pharmacokinetics, with decreased levels of cocaine measured in the brain of immunized mice only 30 seconds after intravenous (i.v.) administration of cocaine. These data establish the feasibility of a therapeutic cocaine vaccine for the treatment of cocaine addiction.

Assessment of ability of murine and human anti-lipid A monoclonal antibodies to bind and neutralize lipopolysaccharide.

The use of monoclonal antibodies (mAbs) directed to lipid A for the therapy of gram-negative sepsis is controversial. In an attempt to understand their biologic basis of action, we used a fluid-phase radioimmunoassay to measure binding between bacterial lipopolysaccharide (LPS) and two IgM mAbs directed to lipid A that are being evaluated for the treatment of gram-negative bacterial sepsis. Both antibodies bound 3H-LPS prepared from multiple strains of gram-negative bacteria when large excesses of antibody were used, although binding was modest and only slightly greater than control preparations. We also studied the ability of each anti-lipid A antibody to neutralize some of the biological effects of LPS in vitro. Despite large molar excesses, neither antibody neutralized LPS as assessed by the limulus lysate test, by a mitogenic assay for murine splenocytes, or by the production of cytokines interleukin (IL)-1, IL-6, or tumor necrosis factor from human monocytes in culture medium or in whole blood. Our experiments do not support the hypothesis that either of these anti-lipid A mAbs function by neutralizing the toxic effects of LPS.

Protective effects of anti-O polysaccharide and anti-lipid A monoclonal antibodies on pulmonary hemodynamics.

Monoclonal antibodies (MAbs) directed to endotoxin can protect in some animal models against the pathophysiological effects of endotoxin infusion. When 0.02 microgram/kg of lipopolysaccharide (LPS) derived from Escherichia coli O111:B4 was incubated in vitro for 2 h with the murine immunoglobulin G MAb, 5B10, directed against the O-polysaccharide antigenic domain of E. coli O111:B4 and then the mixture was infused into sheep, we noted significant protection. The second temperature peak was decreased (P < 0.05 vs. LPS control). The acute pulmonary arterial pressure elevation was diminished (mean peak pulmonary arterial pressure 23.2 +/- 2.5 mmHg, P < 0.05 vs. LPS control), and the peak plasma thromboxane B2 level was reduced (mean peak thromboxane B2 level 0.50 +/- 0.15 ng/ml, P < 0.05 vs. LPS control). In contrast, preincubation of the LPS with a human immunoglobulin M MAb, HA-1A, directed against the core glycolipid of the LPS molecule provided no protective effects in this sheep model. This finding is in agreement with recent studies reporting HA-1A may bind to antibiotic-treated bacteria but not to purified smooth LPS.

The influence of different sitting positions on cervical and lumbar posture.

STUDY DESIGN: This study used a repeated measures design to assess the effects of multiple sitting postures on various spinal angles. All subjects were tested in slouched, erect, forward inclined, and comfortable postures. OBJECTIVES: The purposes of this study were to evaluate the changes in head, cervical, lumbar, and pelvic postures in different sitting positions and also to determine if there is a relation between lumbar posture and cervical posture during sitting. SUMMARY OF BACKGROUND DATA: Clinicians commonly assert that head and neck position is strongly influenced by lumbar and pelvic position. A biomechanical model was developed that allowed detailed, quantitative description of head, neck, lumbar, and pelvic postures. This model enabled a distinction to be made between upper and lower cervical motions. METHODS: Various spinal angles were measured in 30 healthy subjects in four sitting positions using a three-dimensional digitizing system. Reliability of the measurement procedure was determined using an intraclass correlation coefficient and the values for most angles was above 0.8. RESULTS: With the exception of head orientation, analysis of variance revealed significant differences in spinal angles between different sitting positions. Head orientation appeared to be maintained by compensatory adjustments in both the upper and lower cervical spine and changes in lumbar posture were associated with compensatory changes in overall cervical position. As the lumbar spine moved toward extension, the cervical spine flexed and as the lumbar spine flexed the cervical spine extended. However, there was variation among subjects as to whether cervical spine adjustments occurred primarily in the upper or lower cervical region. CONCLUSIONS: Different sitting postures clearly resulted in changes in cervical spine position. Lumbar and pelvic position should be considered when control of cervical posture is desired.

Salivary gland tumors.

A retrospective review of 643 patients with salivary gland tumors seen between 1958-72 is reported. There were 328 malignant and 375 benign tumors. All patients with malignant tumors were assessed in a multidisciplinary head and neck clinic. The median age for developing malignant tumors was 58 and there was a male to female ratio of 1.2:1. For benign tumors the median age was 46 years and the male to female ratio 0.8:1. Overall the primary tumor was controlled by the first planned treatment in 145 (44%) malignant tumors and in 253 (80%) benign tumors. The five and 10 year actuarial survival for malignant tumors was 59.4% and 45.6% respectively.

Adenoid cystic carcinoma of the salivary glands.

Among 334 malignant tumours of the salivary glands 62 (18.6%) were adenoid cystic carcinomas. Although this tumour grows slowly, local recurrence and metastatic spread, particularly to lung, are common. The recommended treatment is surgical excision followed by radiotherapy. Postoperative irradiation reduces the frequency of local recurrence caused by residual microscopic disease. The 5- to 10-year actuarial survival rates were 77% and 57%, respectively. Local and regional recurrence can be eradicated by aggressive treatment, either by radical operation or a combination of operation and irradiation.

Conserved tryptophan in cytochrome c: importance of the unique side-chain features of the indole moiety.

The absolute conservation of tryptophan at position 59 in cytochrome c is related to the unique chemical nature of its indole moiety. The indole side chain of Trp-59 possesses three salient features: bulk, hydrophobicity and the ability of its indole nitrogen to act as a hydrogen-bond donor. Crystallographic evidence identifies the indole nitrogen of Trp-59 as having a stabilizing hydrogen-bonding interaction with the buried carboxylate group of haem propionate 7. Side-chain bulk is also likely to be important because a Phe or Leu residue can replace Trp to give an at least partly functional protein, whereas the smaller Gly or Ser cannot. Semisynthetic analogues were designed to test the importance of the side-chain features of tryptophan by using a recently developed method for stereoselective fragment religation in yeast cytochrome c. Three yeast iso-1 cytochrome c analogues were produced in which Trp-59 was replaced by a non-coded amino acid: p-iodophenylalanine, beta-(3-pyridyl)-alanine or beta-(2-naphthyl)-alanine. Replacement of Trp-59 with these non-coded amino acids allows the reasons for its conservation to be analysed, because they vary with respect to size, hydrophobicity and hydrogen-bond potential. Our results show that decreasing the bulk and hydrophobicity of the side chain at position 59 has a profound but different impact on physicochemical and biological parameters from those of abolishing hydrogen-bond donor potential. This suggests that Trp-59 has both a local and a global stability effect by solvating a buried charge and by having a key role in the packing of the cytochrome c hydrophobic core.

Cytokine priming of human basophils: description of allergen 'nonreleasers'.

Interleukins 3 and 5 and GM-CSF enhance histamine release from basophils triggered by various stimuli. In this report, we describe a subset of allergic patients whose basophils release histamine in response to allergen only when primed with cytokine. In the absence of cytokine, there is no detectable response to allergen. These patients, who represent 4-13% of the allergic population, cannot be distinguished by skin test reactivity or severity of allergic symptoms. Allergen nonreleasers tend to have lower titers of allergen-specific IgE than the majority of atopic subjects, but this difference is not significant (average titer of 29.8 for nonreleasers vs. 188 for typical allergies; p = 0.15). They release histamine normally with anti-IgE and with fMLP, indicating that basophils are responsive to signalling through the IgE receptor, and there is no intrinsic defect in degranulation. Thus, in these patients, the IgE-mediated release of inflammatory mediators from basophils is dependent on, rather than merely enhanced by, T cell cytokines. The relationship between these patients and the previously described anti-IgE 'nonreleasers' is discussed.

Synthetic peptides that mimic the binding site of horseshoe crab antilipopolysaccharide factor.

Tachypleus antilipopolysaccharide (LPS) factor (TALF) is a protein of 102 amino acids in the lysate of amebocytes of Tachypleus tridentatus that binds bacterial LPS with high affinity and blocks its biologic activity in numerous assays. To elucidate the minimal sequences that bind LPS, overlapping synthetic peptides based on the sequence of TALF were assessed for the ability to bind and neutralize LPS. TALF41-53 was the minimal sequence that bound LPS, as assessed by a slot blot capture assay. TALF29-59 bound LPS with the highest potency. TALF29-59 decreased LPS-induced coagulation of limulus amebocyte lysate, induction of cytokines from human monocytes, and LPS-induced lethality in sensitized mice. Synthetic peptides based on TALF or other LPS-binding proteins may be useful for the design of drugs for treatment of endotoxemia.

Verbal recall and recognition following traumatic brain injury: a [0-15]-water positron emission tomography study.

Although several studies exist which have examined static functional neuroimaging following traumatic brain injury (TBI), controlled cognitive activation studies of episodic memory in this population have not been published. The present investigation studied verbal recall using [O-15]-water positron emission tomography (PET) in 5 individuals who sustained severe TBI (M GCS=6.8; M years post-injury=3.18), and 4 non-injured control participants. Statistical image analysis demonstrated changes in frontoparietal regional cerebral blood flow (rCBF) in both groups, but there were interesting differences between groups and across conditions. Frontal lobe rCBF changes in TBI patients were reduced during free recall but enhanced during recognition, when compared to controls. Changes in cerebellar rCBF were observed in the control group during free recall, but not in the TBI sample. In both groups, bifrontal rCBF increases were noted on recognition tasks. The present findings provide evidence of alterations in specific substrates involved in verbal recall following brain injury.

Antiserum against Escherichia coli J5 contains antibodies reactive with outer membrane proteins of heterologous gram-negative bacteria.

The binding of IgG in antiserum to Escherichia coli J5 to the surface of Enterobacteriaceae and to cell wall fragments released from serum-exposed bacteria was studied in a search for potentially protective epitopes other than lipopolysaccharide (LPS). IgG titers to multiple heterologous gram-negative smooth bacteria increased following incubation of the bacteria in serum and decreased following absorption with serum-exposed heterologous bacteria. IgG eluted from absorbing bacteria bound to at least three conserved bacterial outer membrane proteins (OMPs), but not LPS, as assessed by immunoblotting. The same OMPs were present in LPS-containing macromolecular cell wall fragments released by incubation of heterologous gram-negative bacteria in human serum. Part of the protection offered by J5 antiserum could be from binding of IgG to conserved OMPs at the bacterial surface or to OMPs in cell-wall fragments released from dying bacteria.

Binding and neutralization of endotoxin by Limulus antilipopolysaccharide factor.

In order to examine the ability of Limulus antilipopolysaccharide factor (LALF) to bind lipopolysaccharide (LPS), we purified LALF to homogeneity from Limulus amoebocyte lysate and coupled it covalently to agarose beads. LALF-coupled beads captured more tritiated LPS from rough and smooth strains of gram-negative bacteria than did control human serum albumin-coupled beads. Unlabeled homologous and heterologous LPS competed for the binding of 3H-LPS to LALF-coupled beads. LALF bound LPS in a dose-dependent manner as assessed by the precipitation of LPS-LALF complexes with 50% saturated ammonium sulfate. We also studied the ability of LALF to neutralize LPS. LPS preincubated with LALF was less mitogenic for murine splenocytes, was less pyrogenic in the rabbit fever assay, was less lethal in mice which had been sensitized to LPS with actinomycin D, and induced less fever, neutropenia, and pulmonary hypertension when infused into sheep. Our findings extend prior studies which suggested that LALF binds to and neutralizes LPS from multiple strains of gram-negative bacteria.