Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis

Formation of lymphatic metastasis is the initial step of generalized spreading of tumor cells and predicts poor clinical prognosis. Lymphatic vessels generally arise within the peritumoral stroma, although the lymphangiopoietic vascular endothelial growth factors (VEGF)-C and -D are produced by tumor cells. In a carefully selected collection of human cervical cancers (stage pT1b1) we demonstrate by quantitative immunohistochemistry and in situ hybridization that density of lymphatic microvessels is significantly increased in peritumoral stroma, and that a subset of stromal cells express large amounts of VEGF-C and VEGF-D. The density of cells producing these vascular growth factors correlates with peritumoral inflammatory stroma reaction, lymphatic microvessel density, and indirectly with peritumoral carcinomatous lymphangiosis and frequency of lymph node metastasis. The VEGF-C- and VEGF-D-producing stroma cells were identified in situ as a subset of activated tumor-associated macrophages (TAMs) by expression of a panel of macrophage-specific markers, including CD68, CD23, and CD14. These TAMs also expressed the VEGF-C- and VEGF-D-specific tyrosine kinase receptor VEGFR-3. As TAMs are derived from monocytes in the circulation, a search in peripheral blood for candidate precursors of VEGFR-3-expressing TAMs revealed a subfraction of CD14-positive, VEGFR-3-expressing monocytes, that, however, failed to express VEGF-C and VEGF-D. Only after in vitro incubation with tumor necrosis factor-alpha, lipopolysaccharide, or VEGF-D did these monocytes start to synthesize VEGF-C de novo. In conclusion VEGF-C-expressing TAMs play a novel role in peritumoral lymphangiogenesis and subsequent dissemination in human cancer.     

Development of a real-time fluorescence PCR assay for rapid detection of the diphtheria toxin gene

We developed and evaluated a real-time fluorescence PCR assay for detecting the A and B subunits of diphtheria toxin (tox) gene. When 23 toxigenic Corynebacterium diphtheriae strains, 9 nontoxigenic C. diphtheriae strains, and 44 strains representing the diversity of pathogens and normal respiratory flora were tested, this real-time PCR assay exhibited 100% sensitivity and specificity. It allowed for the detection of both subunits of the tox gene at 750 times greater sensitivity (2 CFU) than the standard PCR (1,500 CFU). When used directly on specimens collected from patients with clinical diphtheria, one or both subunits of the tox gene were detected in 34 of 36 specimens by using the real-time PCR assay; only 9 specimens were found to be positive by standard PCR. Reamplification by standard PCR and DNA sequencing of the amplification product confirmed all real-time PCR tox-positive reactions. This real-time PCR format is a more sensitive and rapid alternative to standard PCR for detection of the tox gene in clinical material.     

Cyst-like cerebral lesions in tuberous sclerosis

Known brain manifestations of tuberous sclerosis (TSC) are cortical sclerotic tubera, giant cell astrocytomas, subependymal calcified nodules in the lateral walls of the lateral ventricles, and white matter heterotopias. In addition, small cyst-like lesions in the white matter have been described. We report on three TSC patients with hitherto undescribed large cyst-like cerebral lesions in subcortical and white matter locations. We emphasize that cystoid brain degeneration is a rare but typical cerebral manifestation of TSC and suggest that, in patients with such lesions, TSC should be taken into consideration.     

The expression and regulation of class II antigens in normal and inflammatory bowel disease peripheral blood monocytes and intestinal epithelium

Elevated constitutive expression of major histocompatibility (MHC) class II antigens occurs in the enterocytes of patients with IBD. It has been suggested that this aberrant expression of class II molecules may play a role in the pathogenesis of IBD. We examined two possible reasons for such a finding. 1) Heightened sensitivity of IBD enterocytes to endogenous gamma interferon (gamma IFN) and 2) enhanced endogenous secretion of gamma interferon by intestinal cells in close proximity to the enterocytes (lamina propria lymphocytes). Constitutive and gamma interferon stimulated HLA-DR and DP density on intestinal epithelial cells (IEC) and peripheral blood monocytes (PBM) from UC patients (IEC n = 13; PBM n = 20), CD patients (IEC n = 14; PBM n = 18) and non-IBD controls (IEC n = 12; PBM n = 20) were measured via flow cytometry (mean channel fluorescence). gamma IFN production by PHA stimulated and unstimulated lamina propria lymphocyte (LPL) cultures of UC patients (n = 11) CD patients (n = 8) and non-IBD controls (n = 11) was measured using a vesicular stomatitis virus/WISH cell bioassay. We found significantly greater gamma IFN secretion by IBD-derived PHA stimulated LPL than from non-IBD stimulated controls (CD = 39.4 +/- 12.4u; UC41.5 +/- 6.8u; NL = 22.4 +/- 8.3u, p less than 0.05) while gamma IFN induced HLA-DR and DP upregulation was no greater in IBD-derived IEC and PBM than in non-IBD controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical, genetic, and epidemiologic characterization of Haemophilus influenzae biogroup aegyptius (Haemophilus aegyptius) strains associated with Brazilian purpuric fever.

Brazilian purpuric fever (BPF) is a recently recognized fulminant pediatric disease characterized by fever, with rapid progression to purpura, hypotensive shock, and death. BPF is usually preceded by purulent conjunctivitis that has resolved before the onset of fever. Both the conjunctivitis and BPF are caused by Haemophilus influenzae biogroup aegyptius (formerly called H. aegyptius). Isolates from 15 BPF cases, mainly from blood or hemorrhagic cerebrospinal fluid, case-associated isolates from 42 persons in towns where BPF cases occurred, and control strains from 32 persons in towns without BPF cases were characterized biochemically, genetically, and epidemiologically. Results indicated that a single clone was responsible for all BPF cases identified in six Brazilian towns from 1984 through 1986. All of 15 (100%) case strains were the same clone as was 1 of 32 (3%) control strains (P = less than 10(-8). Isolates of the clone were preferentially intrarelated by DNA hybridization (99% relatedness, hydroxyapatite method at 60 and 75 degrees C) and were separable from other H. influenzae biogroup aegyptius strains (approximately 90% relatedness at 60 degrees C and 82% relatedness at 75 degrees C). All isolates of the BPF clone and no other strains contained a 24-megadalton plasmid of restriction endonuclease type 3031, were of a single multilocus enzyme mobility type, were of a single sodium dodecyl sulfate-polyacrylamide gel electrophoresis type, and were in one of two ribosomal DNA restriction patterns. All BPF clone isolates reacted with monoclonal antibodies produced from a case strain; only 3 of 62 (5%) other strains reacted with this monoclonal antibody. Ninety percent of BPF clone strains and 27% of other strains were relatively resistant to sulfamethoxazole-trimethoprim.     

Sites of antagonist action on N-methyl-D-aspartic acid receptors studied using fluctuation analysis and a rapid perfusion technique

1. Mouse hippocampal neurons in dissociated culture were grown at low density on previously plated hippocampal glial cell cultures and voltage clamped using the tight seal whole-cell patch-clamp technique. Flow pipes were used to rapidly exchange the extracellular solution, and to apply N-methyl-D-aspartic acid (NMDA) and some NMDA antagonists. Fluctuation analysis was used to estimate changes in the behavior of NMDA-activated ion channels during application of antagonists. In the presence of NMDA control spectra were well fit by single Lorentzian functions consistent with mean open times of 5-6 ms. 2. Two antagonists thought to act at the NMDA receptor agonist recognition site, 2-amino-5-phosphonovaleric acid (AP5) and kynurenic acid, did not produce changes in the mean open time or single channel conductance, consistent with their action as competitive antagonists. Onset of antagonism and recovery from the action of both AP5 and kynurenic acid was rapid and complete within 1 s. However, raising the extra-cellular glycine concentration, from 1 microM to 1 mM, reduced the potency of 100 microM kynurenic acid as an NMDA antagonist, suggesting that kynurenate has an additional action as a competitive antagonist at the glycine modulatory site on NMDA receptor channels. 3. In the presence of 150 microM magnesium NMDA spectra recorded at -60 mV were fit by double Lorentzian functions, consistent with single-channel events consisting of bursts of openings lasting 3.3 ms in duration, interrupted by blocking and unblocking events of average duration 0.18 ms. The onset and recovery from magnesium antagonism was rapid, and complete within 1 s, but was highly voltage dependent and at +40 mV magnesium (150 microM) failed to produce NMDA antagonism. These results are consistent with a voltage-dependent channel block of NMDA receptor channels produced by binding of magnesium to a site within the ion channel. 4. Zinc (30 microM) was a potent NMDA antagonist at both -60 and +40 mV, and at either potential appeared to reduce the mean open time of NMDA-activated ion channels from about 5 ms to approximately 3 ms. Over the frequency range measured, 1-1,000 Hz, NMDA spectra were well fit by single Lorentzians during zinc antagonism, in contrast to results obtained with magnesium. The mean single channel conductance also decreased in the presence of zinc to approximately 75% of control. Onset of antagonism and recovery from the action of zinc was rapid and complete within 1 s.(ABSTRACT TRUNCATED AT 400 WORDS)     

An open,randomized single-centre study to compare the efficacy and convenience of follitropin beta administered by a pen device with follitropin alpha administered by a conventional syringe in women undergoing ovarian stimulation for IVF/ICSI

BACKGROUND: A pen device, similar to an insulin pen, has been recently marketed for the administration of follitropin beta in cartridges. A randomized controlled trial was performed to compare the efficacy and convenience of this pen device delivering follitropin beta with a conventional syringe delivering follitropin alpha. METHODS: A total of 200 patients needing IVF/ICSI treatment and willing to self-inject were enrolled in the study. All subjects had ovarian stimulation according to a long protocol and were randomized to the pen or the conventional syringe group during down-regulation by means of a computer-generated randomization list using random numbers. Patients were asked to fill in a daily local tolerance book after each injection. On the day of hCG the patients scored a Visual Analogue Scale (VAS) for pain and convenience. RESULTS: The average duration, total dose of recombinant FSH and number of cumulus oocyte complexes retrieved were 10.8/12.0 days (P = 0.001), 1880/2226 IU (P < 0.001) and 15.2/13.1 respectively in the pen device and conventional syringe groups; the presence of pain after the daily injection was significantly higher in the conventional syringe group (P = 0.027); the visual analogue scale score was similar for pain but significantly more convenient for the pen device (P < 0.001). The live birth rate per embryo transfer was 32.9 and 34.4% respectively in the pen device and conventional syringe groups. CONCLUSIONS: Self-injection with the pen device is safe and easy, more convenient and less painful for the patient, requires less FSH and shortens the treatment duration.     

Comparison of four different methods for detection of rubella IgM antibodies

Four different tests for detection of rubella-specific IgM antibodies were compared: two Ig separation methods (centrifugation and chromatography) with subsequent haemagglutination inhibition test and two commercially available ELISA tests. The 114 sera tested had been sent to the diagnostic laboratory, mostly with insufficient clinical histories. Agreement between the centrifugation method and one of the ELISA tests was good (2 divergent results with 107 sera tested), while the other ELISA test yielded more positive (partly perhaps non-specific) results. The chromatographic method did not separate the Ig classes as reliably as the centrifugation method, but because of its simplicity it may be useful, if adequate test controls are performed. The divergent results are discussed. It is postulated that in cases with pending induced abortion, two independent tests should be performed.