Protection against ischemic brain damage using propentofylline in gerbils

We studied the xanthine derivative propentofylline (HWA 285) to determine its protection against ischemic brain damage when administered before and after ischemia. Transient forebrain ischemia was produced in 81 Mongolian gerbils by occluding both carotid arteries. The necessary surgery was performed under anesthesia with intraperitoneal pentobarbital/chloral hydrate 2 days before occlusion. We tested the parameters delayed selective hippocampal nerve cell damage, generation of seizures, and survival. Determination of the dose-response relation revealed the optimal dose of HWA 285 to be 10 mg/kg i.p. The effect of the drug on delayed selective nerve cell damage in the hippocampus was assessed by measuring the intensity of Nissl staining in the CA1 area by means of densitometry 4 days after a 10-minute occlusion. Gerbils treated with HWA 285 revealed significant protection of the CA1 neurons even when the drug was applied 1 hour after the end of the occlusion. In contrast to HWA 285, pentobarbital provided no detectable protection of the CA1 neurons in our experimental model when administered 1 hour after occlusion, suggesting different mechanisms of action. After a 15-minute occlusion, all six untreated gerbils developed convulsions and died within 2 days. Chronic (daily) treatment of nine gerbils with HWA 285 prevented the generation of convulsions in eight and allowed seven to survive for greater than 12 days.     

Hydroa vacciniforme

Two patients with hydroa vacciniforme, a rare photodermatosis of unknown etiology, demonstrated distinctive scarring and vesiculobullous skin lesions on light-exposed body areas. Results of blood and urine porphyrin studies were normal, and no systemic abnormalities were noted. A small bullous lesion was produced in normal skin in case 1 with 15 times the minimal erythema dose of ultraviolet energy. The conditions of both patients improved while they were taking beta carotene orally.     

Cell surface antigens of chemically induced sarcomas of murine origin

Chemically induced sarcomas of murine origin are characterized by the expression of unique tumour-specific transplantation antigens. Their nature and the genetic basis of their antigenic diversity are unknown. Numerous attempts have been made to relate these antigens to products of known polymorphic gene families. A serological analysis of cell-surface antigens of sarcomas was initiated on the premise that serologically defined unique tumour-specific antigens would be related to tumour-specific transplantation antigens. This analysis led to the serological definition of two noncross-reacting tumour-specific antigens on two antigenically distinct BALB/c sarcomas, Meth A and CMS4. Assignment of the gene(s) involved in expression of the Meth A antigen to the distal region of chromosome 12, the same region encoding Igh, raised the possibility that genetic elements responsible for antibody idiotype could be involved in the antigenic diversity of tumour-specific transplantation antigens. Although several studies indicate a concordant expression of the Meth A and the tumour-specific transplantation antigen, the Meth A tumour-specific transplantation antigen identified as an 82,000-Da protein does not express the serologically defined antigen. This is in contrast to the Zajdela hepatoma of rat origin, where a serologically defined tumour-specific antigen is related to the tumour-specific transplantation antigen. Whether the serologically defined Meth A antigen has tumour-specific transplantation antigen activity is not presently known.     

Impact of p53-based immunization on primary chemically-induced tumors

In mice as well as humans, cytotoxic T lymphocytes (CTL) specific for wild-type-sequence (wt) p53 peptides have been shown to react against a wide range of tumors, but not normal cells. As such, they are attractive candidates for developing broadly applicable cancer vaccines. Of particular interest is the potential of using p53-based vaccines in high-risk individuals to prevent cancer. Methylcholanthrene, an immunosuppressive polycyclic hydrocarbon carcinogen implicated as a causative agent in human cancers, has long been used to induce murine tumors with a high incidence of genetic alterations and sensitivity to wt p53-specific CTL. To analyze the potential of p53-based vaccines on primary tumors, we evaluated the efficacy of DNA and dendritic cell vaccines targeting wt p53 peptides given to methylcholanthrene-treated mice in the protection or therapy settings. The results indicate that the efficacy of these vaccines relative to reducing tumor incidence were severely compromised by vaccine-induced tumor escape. As compared to tumors induced in non-immunized mice, a higher incidence of epitope-loss tumors was detected in tumors from the immunized mice. The increase in tumor escape arose as a consequence of either increased frequencies of mutations within/flanking p53 epitope-coding regions or downregulation of expression of the major histocompatibility complex Class I molecules that present these epitopes for T cell recognition These findings are consistent with current views of immunoselection occurring in patients receiving tumor peptide-based immunotherapy, and impact on the design and implementation of p53-based vaccines, in particular, those aimed at treating individuals at high risk for developing cancer.     

Complete nucleotide sequence analysis of plasmids in strains of Staphylococcus aureus clone USA300 reveals a high level of identity among isolates with closely related core genome sequences

A community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain known as pulsed-field type USA300 (USA300) is epidemic in the United States. Previous comparative whole-genome sequencing studies demonstrated that there has been recent clonal emergence of a subset of USA300 isolates, which comprise the epidemic clone. Although the core genomes of these isolates are closely related, the level of diversity among USA300 plasmids was not resolved. Inasmuch as these plasmids might contribute to significant gene diversity among otherwise closely related USA300 isolates, we performed de novo sequencing of endogenous plasmids from 10 previously characterized USA300 clinical isolates obtained from different geographic locations in the United States. All isolates tested contained small (2- to 3-kb) and/or large (27- to 30-kb) plasmids. The large plasmids encoded heavy metal and/or antimicrobial resistance elements, including those that confer resistance to cadmium, bacitracin, macrolides, penicillin, kanamycin, and streptothricin, although all isolates were sensitive to minocycline, doxycycline, trimethoprim-sulfamethoxazole, vancomycin, teicoplanin, and linezolid. One of the USA300 isolates contained an archaic plasmid that encoded staphylococcal enterotoxins R, J, and P. Notably, the large plasmids (27 to 28 kb) from 8 USA300 isolates--those that comprise the epidemic USA300 clone--were virtually identical (99% identity) and similar to a large plasmid from strain USA300_TCH1516 (a previously sequenced USA300 strain from Houston, TX). These plasmids are largely divergent from the 37-kb plasmid of FPR3757, the first sequenced USA300 strain. The high level of plasmid sequence identity among the majority of closely related USA300 isolates is consistent with the recent clonal emergence hypothesis for USA300.     

Progesterone mediates gonadal hormone differences in tactile and thermal hypersensitivity following L5 nerve root ligation in female rats

Sex differences in the magnitude of response to thermal and tactile stimuli have been demonstrated in both clinical and animal studies. Female rats typically display lower thresholds to painful stimuli and display more robust responses following nerve injury as compared with males. There is a body of evidence implicating the sex hormones in mediating this sex difference. In the present study, we sought to determine which gonadal hormones were involved in mediating the observed female hypersensitivity in female rats both prior to and following experimental nerve root injury using a chronic hormone replacement paradigm. Female rats were ovariectomized and hormone pellets containing 17beta-estradiol, progesterone (P), 17beta-estradiol+progesterone or placebo were implanted s.c. Our results demonstrate that only the group of female rats that received progesterone alone maintained the hypersensitive phenotype following ovariectomy, compared with gonadally intact male rats. This result was observed both in response to thermal stimuli in non-injured female rats and to thermal and tactile stimuli following L5 nerve root ligation, a model of low back pain associated with lumbar radiculopathy. Postmortem analysis of serum gonadal hormone concentrations demonstrates that the hormonal manipulations were successful and the exogenous hormones were similar to physiological levels observed in the sham-ovariectomized controls. Taken together, these results demonstrate the critical role for progesterone in mediating enhanced female tactile and thermal hypersensitivity following L5 nerve root ligation.     

Molecular differentiation of historic phage-type 80/81 and contemporary epidemic Staphylococcus aureus

Staphylococcus aureus is a bacterial pathogen known to cause infections in epidemic waves. One such epidemic was caused by a clone known as phage-type 80/81, a penicillin-resistant strain that rose to world prominence in the late 1950s. The molecular underpinnings of the phage-type 80/81 outbreak have remained unknown for decades, nor is it understood why related S. aureus clones became epidemic in hospitals in the early 1990s. To better understand the molecular basis of these epidemics, we sequenced the genomes of eight S. aureus clinical isolates representative of the phage-type 80/81 clone, the Southwest Pacific clone [a community-associated methicillin-resistant S. aureus (MRSA) clone], and contemporary S. aureus clones, all of which are genetically related and belong to the same clonal complex (CC30). Genome sequence analysis revealed that there was coincident divergence of these clones from a recent common ancestor, a finding that resolves controversy about the evolutionary history of the lineage. Notably, we identified nonsynonymous SNPs in genes encoding accessory gene regulator C (agrC) and α-hemolysin (hla)--molecules important for S. aureus virulence--that were present in virtually all contemporary CC30 hospital isolates tested. Compared with the phage-type 80/81 and Southwest Pacific clones, contemporary CC30 hospital isolates had reduced virulence in mouse infection models, the result of SNPs in agrC and hla. We conclude that agr and hla (along with penicillin resistance) were essential for world dominance of phage-type 80/81 S. aureus, whereas key SNPs in contemporary CC30 clones restrict these pathogens to hospital settings in which the host is typically compromised.     

Acute peripheral inflammation induces moderate glial activation and spinal IL-1beta expression that correlates with pain behavior in the rat.

Our laboratory has previously shown that glial activation and increased proinflammatory cytokine expression are observed in the rat spinal cord following peripheral nerve injuries that result in neuropathic pain behaviors. In the present study, we sought to determine whether acute peripheral inflammation induces changes in central glial and cytokine (Interleukin-1beta) expression similar to those seen following peripheral spinal nerve transection. Two models of peripheral inflammation were used in this study: formalin (5% solution) or zymosan (25 mg/ml) injected subcutaneously into the plantar portion of the left hind paw of male Holtzman-strain Sprague-Dawley rats. The rats were euthanized at 1 h, 6 h, and 1, 3, 7 days post-injection (n=4 or 5/group/time point). As expected, the animals treated with formalin showed a spontaneous pain response and mechanical allodynia that persisted for approximately 60 min following injection. The animals treated with zymosan exhibited mild spontaneous pain responses during the first hour and mechanical allodynia at 6 h and 1 day following injection. Immunohistochemistry for glial activation and cytokine expression was performed on L4-L5 spinal levels in all rats. Spinal sections from both formalin and zymosan treated animals exhibited microglial and astrocytic activation and increased Interleukin-1beta immunoreactivity at 1 and 6 h, respectively. Spinal glial activation and upregulation of Interleukin-1beta appear to parallel the development and maintenance of zymosan and formalin-induced mechanical allodynia. These findings support a unifying theory that glial activation and cytokine expression have a similar, if not related, role in producing hyperalgesia following either peripheral inflammation or peripheral nerve injury.     

Targeted signal-amplifying enzymes enhance MRI of EGFR expression in an orthotopic model of human glioma

Epidermal growth factor receptor (EGFR) imaging in brain tumors is essential to visualize overexpression of EGFRvIII variants as a signature of highly aggressive gliomas and to identify patients that would benefit from anti-EGFR therapy. Seeking imaging improvements, we tested a novel pretargeting approach that relies on initial administration of enzyme-linked anti-EGFR monoclonal antibodies (mAb; EMD72000) followed by administration of a low-molecular-weight paramagnetic molecule (diTyr-GdDTPA) retained at the site of EGFR mAb accumulation. We hypothesized that diTyr-GdDTPA would become enzyme activated and retained on cells due to binding to tissue proteins. In support of this hypothesis, mAb-enzyme conjugates reacted with both membrane-isolated wild-type (wt) EGFR and EGFRvIII, but they bound primarily to EGFRvIII-expressing cells and not to EGFRwt-expressing cells. In vivo analysis of magnetic resonance (MR) tumor signal revealed differences in MR signal decay following diTyr-GdDTPA substrate administration. These differences were significant in that they suggested differences in substrate elimination from the tissue which relied on the specificity of the initial mAb binding: a biexponential signal decay was observed in tumors only upon preinjection with EGFR-targeted conjugates. Endpoint MRI in this setting revealed detailed images of tumors which correlated with immunohistochemical detection of EGFR expression. Together, our findings suggest an improved method to identify EGFRvIII-expressing gliomas in vivo that are best suited for treatment with therapeutic EGFR antibodies.