Image analysis derived ploidy and proliferation indices in soft tissue sarcomas: comparison with clinical outcome.

AIMS--To compare prognostic information obtained by image analysis cytometry of paraffin wax embedded soft tissue sarcomas with conventional assessment. METHODS--A CAS 200 image analyser was used to determine DNA content of Feulgen stained cytology preparations and tissue sections and to quantify immunostaining by Ki67 and PC10 antibodies. A mitotic count in 50 high power fields was undertaken and histological grade assigned by the Trojani system. Clinical details including follow up and outcome were obtained by case note review. The Kruskal-Wallis one way analysis test, Spearman rho significance test, Kaplan-Meier method, and log-rank test were applied in statistical analysis. RESULTS--Ploidy status, DNA index, 2.5c exceeding rate, 5c exceeding rate, mitotic count and Trojani grade all correlated significantly with clinical outcome. The relation between Ki67 index and outcome did not reach significance. The PC10 index and outcome were not related. Only 2.5c exceeding rate, 5c exceeding rate, and mitotic count correlated significantly with Trojani grade. CONCLUSIONS--DNA content determination of soft tissue sarcomas by image analysis provides quantifiable information of benefit in prediction of outcome. Larger series are required to determine the independent value of ploidy. In this study quantification of anti-Ki67 and anti-PC10 immunostaining was not of prognostic benefit) by contrast with mitotic count and Trojani grade.     

Volunteer studies of deletion mutants of Vibrio cholerae O1 prepared by recombinant techniques

Vibrio cholerae O1 A-B- vaccine strain JBK 70 and A-B+ CVD 101 prepared by recombinant DNA techniques from pathogenic EI Tor Inaba N16961 and classical Ogawa 395, respectively, were fed to 38 volunteers in single doses of 10(4) to 10(10). Although severe diarrhea did not occur in any vaccine, more than one-half developed mild diarrhea. These attenuated strains colonized well and elicited prominent vibriocidal and antitoxic (CVD 101) antibody responses. Recipients of a single dose of JBK 70 were significantly protected when challenged with 10(6) wild-type N16961. Diarrhea occurred in 7 of 8 controls but in only 1 of 10 vaccines (P less than 0.003, 89% vaccine efficacy), demonstrating the potency of immune mechanisms that do not involve cholera antitoxin. Further derivatives were prepared to explore the pathogenesis of the residual diarrhea, considering that either intestinal colonization by the vaccine itself or accessory toxins might be responsible. CVD 102, an auxotrophic mutant of CVD 101, did not cause diarrhea but colonized poorly and elicited feeble immune responses. Derivatives of JBK 70 and CVD 101 (CVD 104 and 105) deleted of genes encoding the EI Tor hemolysin still caused mild diarrhea. Genetically engineered strains can be colonizing, highly immunogenic, and protective single-dose oral vaccines, but they must be further attenuated before they can be considered for use as public health tools.     

The role of post-synaptic neurones in the biochemical maturation of presynaptic cholinergic nerve terminals in a mouse sympathetic ganglion

1. The role of post-synaptic adrenergic neurones in the biochemical maturation of presynaptic cholinergic nerve terminals has been investigated in mouse superior cervical ganglion in vivo.2. Selective destruction of ganglion adrenergic neurones chemically, with 6-hydroxydopamine (6-OH-DA), or immunologically, with nerve growth factor antiserum (NGF-antiserum) prevented the normal maturation of choline acetyl transferase (ChAc) activity in presynaptic endings during development. Enzyme activity remained depressed for at least 2 months.3. 6-OH-DA treatment failed to alter ChAc activity in the developing duodenum or diaphragm, organs in which cholinergic fibres do not synapse with adrenergic neurones, suggesting that destruction of post-synaptic neurones per se inhibited presynaptic maturation.4. Similarly, NGF-antiserum, which does not destroy adrenergic neurones in the adult did not alter ChAc activity in adult mouse ganglia.5. These observations suggest that post-synaptic adrenergic neurones regulate the biochemical development of presynaptic cholinergic nerve terminals.     

In vivo NGF deprivation reduces SNS expression and TTX-R sodium currents in IB4-negative DRG neurons

Recent evidence suggests that changes in sodium channel expression and localization may be involved in some pathological pain syndromes. SNS, a tetrodotoxin-resistant (TTX-R) sodium channel, is preferentially expressed in small dorsal root ganglion (DRG) neurons, many of which are nociceptive. TTX-R sodium currents and SNS mRNA expression have been shown to be modulated by nerve growth factor (NGF) in vitro and in vivo. To determine whether SNS expression and TTX-R currents in DRG neurons are affected by reduced levels of systemic NGF, we immunized adult rats with NGF, which causes thermal hypoalgesia in rats with high antibody titers to NGF. DRG neurons cultured from rats with high antibody titers to NGF, which do not bind the isolectin IB4 (IB4(-)) but do express TrkA, were studied with whole cell patch-clamp and in situ hybridization. Mean TTX-R sodium current density was decreased from 504 +/- 77 pA/pF to 307 +/- 61 pA/pF in control versus NGF-deprived neurons, respectively. In comparison, the mean TTX-sensitive sodium current density was not significantly different between control and NGF-deprived neurons. Quantification of SNS mRNA hybridization signal showed a significant decrease in the signal in NGF-deprived neurons compared with the control neurons. The data suggest that NGF has a major role in the maintenance of steady-state levels of TTX-R sodium currents and SNS mRNA in IB4(-) DRG neurons in adult rats in vivo.     

Gamma interferon responses induced by a panel of recombinant and purified mycobacterial antigens in healthy,non-mycobacterium bovis BCG-vaccinated Malawian young adults

We have previously shown that young adults living in a rural area of northern Malawi showed greater gamma interferon (IFN-gamma) responses to purified protein derivatives (PPD) prepared from environmental mycobacteria than to PPD from Mycobacterium tuberculosis. In order to define the mycobacterial species to which individuals living in a rural African population have been exposed and sensitized, we tested T-cell recognition of recombinant and purified antigens from M. tuberculosis (38 kDa, MPT64, and ESAT-6), M. bovis (MPB70), M. bovis BCG (Ag85), and M. leprae (65 kDa, 35 kDa, and 18 kDa) in >600 non-M. bovis BCG-vaccinated young adults in the Karonga District of northern Malawi. IFN-gamma was measured by enzyme-linked immunosorbent assay (ELISA) in day 6 supernatants of diluted whole-blood cultures. The recombinant M. leprae 35-kDa and 18-kDa and purified native M. bovis BCG Ag85 antigens induced the highest percentages of responders, though both leprosy and bovine tuberculosis are now rare in this population. The M. tuberculosis antigens ESAT-6 and MPT64 and the M. bovis antigen MPB70 induced the lowest percentages of responders. One of the subjects subsequently developed extrapulmonary tuberculosis; this individual had a 15-mm-diameter reaction to the Mantoux test and responded to M. tuberculosis PPD, Ag85, MPT64, and ESAT-6 but not to any of the leprosy antigens. We conclude that in this rural African population, exposure to M. tuberculosis or M. bovis is much less frequent than exposure to environmental mycobacteria such as M. avium, which have antigens homologous to the M. leprae 35-kDa and 18-kDa antigens. M. tuberculosis ESAT-6 showed the strongest association with the size of the Mantoux skin test induration, suggesting that among the three M. tuberculosis antigens tested it provided the best indication of exposure to, or infection with, M. tuberculosis.     

Ion channels in spinal cord astrocytes in vitro. I. Transient expression of high levels of Na+ and K+ channels.

1. Na+ and K+ channel expression was studied in cultured astrocytes derived from P--0 rat spinal cord using whole cell patch-clamp recording techniques. Two subtypes of astrocytes, pancake and stellate, were differentiated morphologically. Both astrocyte types showed Na+ channels and up to three forms of K+ channels at certain stages of in vitro development. 2. Both astrocyte types showed pronounced K+ currents immediately after plating. Stellate but not pancake astrocytes additionally showed tetrodotoxin (TTX)-sensitive inward Na+ currents, which displayed properties similar to neuronal Na+ currents. 3. Within 4-5 days in vitro (DIV), pancake astrocytes lost K(+)-current expression almost completely, but acquired Na+ currents in high densities (estimated channel density approximately 2-8 channels/microns2). Na+ channel expression in these astrocytes is approximately 10- to 100-fold higher than previously reported for glial cells. Concomitant with the loss of K+ channels, pancake astrocytes showed significantly depolarized membrane potentials (-28.1 +/- 15.4 mV, mean +/- SD), compared with stellate astrocytes (-62.5 +/- 11.9 mV, mean +/- SD). 4. Pancake astrocytes were capable of generating action-potential (AP)-like responses under current clamp, when clamp potential was more negative than resting potential. Both depolarizing and hyperpolarizing current injections elicited overshooting responses, provided that cells were current clamped to membrane potentials more negative than -70 mV. Anode-break spikes were evoked by large hyperpolarizations (less than -150 mV). AP-like responses in these hyperpolarized astrocytes showed a time course similar to neuronal APs under conditions of low K+ conductance. 5. In stellate astrocytes, AP-like responses were not observed, because the K+ conductance always exceeded Na+ conductance by at least a factor of 3. Thus stellate spinal cord astrocyte membranes are stabilized close to EK as previously reported for hippocampal astrocytes. 6. It is concluded that spinal cord pancake astrocytes are capable of synthesizing Na+ channels at densities that can, under some conditions, support electrogenesis. In vivo, however, AP-like responses are unlikely to occur because the cells' resting potential is too depolarized to allow current activation. Thus the absence of electrogenesis in astrocytes may be explained by two mechanisms: 1) a low Na-to-K conductance ratio, as in stellate spinal cord astrocytes and in other previously studied astrocyte preparations; or, 2) as described in detail in the companion paper, a mismatch between the h infinity curve and resting potential, which results in Na+ current inactivation in spinal cord pancake astrocytes.     

Identification of a nucleocapsid protein as a specific serological marker of human herpesvirus 6 infection.

Enveloped whole virions and nucleocapsids of human herpesvirus 6 (HHV-6) strain Z29 were purified from supernatant fluids of infected human cord blood lymphocytes by filtration through polyvinylpyrrolidone-treated filters, banding on a Nycondenz step gradient, and centrifugation through two successive continuous sucrose gradients. More than 20 proteins ranging in molecular weight from less than 30,000 to more than 200,000 were identified in preparations of purified whole virions labeled with [35S]methionine and [35S]cysteine. Immunogenic virion proteins of HHV-6 were identified in immunoblot assays with human immune sera, immune sera generated from mice immunized with purified whole virions or purified nucleocapsids, and a monoclonal antibody generated from a mouse immunized with purified nucleocapsids. The sera and the monoclonal antibody reacted strongly with a 101-kilodalton protein in the immunoblots, suggesting that the protein is a component of the nucleocapsid. Human sera lacking HHV-6-specific antibodies and seropositive for one or more of the other human herpesviruses failed to react with this protein, indicating that it is a specific serologic marker for HHV-6 infection.     

2-Mercaptoethanol and n-acetylcysteine enhance T cell colony formation in AIDS and ARC.

One contributing factor to the loss of T cells in AIDS may be the impaired ability of T cell precursors to expand, as reflected in a decreased ability of patient cells to form T cell colonies in agar. We and others have noted such a defect in people with AIDS and ARC, and have found that suppressor cells and suppressive plasma contribute to decreased T-CFC formation. We report here that the reducing agents 2-mercaptoethanol (2-ME) and n-acetyl cysteine (NAC) can enhance colony formation in vitro. In part, 2-ME can reverse the defect in T cell colony-forming cells (T-CFC) formation by overcoming the effect of suppressor cells. In a group of 46 AIDS patients, T-CFC formation was initially 42 +/- 8% (mean +/- s.e.) that of control levels. 2-ME caused an increase of 401 +/- 76% in T-CFC formation which was significantly greater than the increase in control T-CFC formation; it also significantly enhanced T-CFC formation by cells from ARC patients. Suppressor cell activity from ten AIDS patients decreased from 58 +/- 21% to 12 +/- 10% when 2-ME was added. Similar data were obtained from 14 ARC patients. NAC, a related antioxidant with low toxicity, also enhanced T-CFC in cells of AIDS and ARC patients. Vitamin C generally did not increase T-CFC formation. The data suggest that certain antioxidants such as 2-ME and NAC may be useful in treatment protocols to enhance T cell numbers in patients with AIDS or ARC.