Mechanisms underlying induced autoresuscitation failure in BALB/c and SWR mice.

Mechanisms underlying failure of autoresuscitation from hypoxic apnea were investigated. Failure was induced by repeated exposure to hypoxia. The influence of maturation was studied in adults, weanlings, and 10- and 5-day-old mice. Mice successful at autoresuscitation (BALB/c) as well as those prone to autoresuscitation failure (SWR weanlings) were studied. Hypoxic apnea was induced with 97% N2-3% CO2, and 21% O2 was given at its onset; electrocardiogram and ventilation were recorded. Hypoxic exposure was repeated if autoresuscitation (recovery of eupnea) occurred. Autoresuscitation failure (death) was induced in all mice. Young BALB/c mice tolerated more trials than older mice. SWR weanlings frequently failed to autoresuscitate on the initial exposure and tolerated fewer repeat trials overall than age-matched BALB/c mice. Induced autoresuscitation failure in all mice appeared to be unrelated to gasping regulation, because both gasp number and amplitude were similar during the failed trial and the previous successful trial. In most mice, failure was associated with absent recovery of heart rate during gasping. In BALB/c mice in particular, this persistent bradycardia was usually due to heart block, which occurred in 95% of failed trials. In addition, heart block occurred with increasing frequency on later successful trials, but conversion to sinus rhythm always preceded successful autoresuscitation. Heart block was also frequent in SWR mice and had similar consequences. BALB/c mice exposed to continuous anoxia survived longer than SWR mice, indicating increased endurance of components of the autoresuscitation mechanism not directly related to the ventilatory function of gasping (e.g., cardiovascular components).(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel family of mobile genetic elements is limited to the germline genome in Tetrahymena thermophila

In the ciliated protozoan Tetrahymena thermophila, extensive DNA elimination is associated with differentiation of the somatic macronucleus from the germline micronucleus. This study describes the isolation and complete characterization of Tlr elements, a family of approximately 30 micronuclear DNA sequences that are efficiently eliminated from the developing macronucleus. The data indicate that Tlr elements are comprised of an ~22 kb internal region flanked by complex and variable termini. The Tlr internal region is highly conserved among family members and contains 15 open reading frames, some of which resemble genes encoded by transposons and viruses. The Tlr termini appear to be long inverted repeats consisting of (i) a variable region containing multiple direct repeats which differ in number and sequence from element to element and (ii) a conserved terminal 47 bp sequence. Taken together, these results suggest that Tlr elements comprise a novel family of mobile genetic elements that are confined to the Tetrahymena germline genome. Possible mechanisms of developmentally programmed Tlr elimination are discussed.     

A family of developmentally excised DNA elements in Tetrahymena is under selective pressure to maintain an open reading frame encoding an integrase-like protein

Tlr1 is a member of a family of ~20–30 DNA elements that undergo developmentally regulated excision during formation of the macronucleus in the ciliated protozoan Tetrahymena. Analysis of sequence internal to the right boundary of Tlr1 revealed the presence of a 2 kb open reading frame (ORF) encoding a deduced protein with similarity to retrotransposon integrases. The ORFs of five unique clones were sequenced. The ORFs have 98% sequence conservation and align without frameshifts, although one has an additional trinucleotide at codon 561. Nucleotide changes among the five clones are highly non-random with respect to the position in the codon and 93% of the nucleotide changes among the five clones encode identical or similar amino acids, suggesting that the ORF has evolved under selective pressure to preserve a functional protein. Nineteen T/C transitions in T/CAA and T/CAG codons suggest selection has occurred in the context of the Tetrahymena genome, where TAA and TAG encode Gln. Similarities between the ORF and those encoding retrotransposon integrases suggest that the Tlr family of elements may encode a polynucleotide transferase. Possible roles for the protein in transposition of the elements within the micronuclear genome and/or their developmentally regulated excision from the macronucleus are discussed.     

Astrocytes Directly Influence Tumor Cell Invasion and Metastasis In Vivo

Brain metastasis is a defining component of tumor pathophysiology, and the underlying mechanisms responsible for this phenomenon are not well understood. Current dogma is that tumor cells stimulate and activate astrocytes, and this mutual relationship is critical for tumor cell sustenance in the brain. Here, we provide evidence that primary rat neonatal and adult astrocytes secrete factors that proactively induced human lung and breast tumor cell invasion and metastasis capabilities. Among which, tumor invasion factors namely matrix metalloprotease-2 (MMP-2) and MMP-9 were partly responsible for the astrocyte media-induced tumor cell invasion. Inhibiting MMPs reduced the ability of tumor cell to migrate and invade in vitro. Further, injection of astrocyte media-conditioned breast cancer cells in mice showed increased invasive activity to the brain and other distant sites. More importantly, blocking the preconditioned tumor cells with broad spectrum MMP inhibitor decreased the invasion and metastasis of the tumor cells, in particular to the brain in vivo. Collectively, our data implicate astrocyte-derived MMP-2 and MMP-9 as critical players that facilitate tumor cell migration and invasion leading to brain metastasis.     

Expression of macrophage migration inhibitory factor by neuroblastoma leads to the inhibition of antitumor T cell reactivity in vivo

Neuroblastomas and many other solid tumors produce high amounts of macrophage migration inhibitory factor (MIF), which appears to play a role in tumor progression. We found that MIF expression in neuroblastoma inhibits T cell proliferation in vitro, raising the possibility that MIF promotes tumorigenesis, in part, by suppressing antitumor immunity. To examine whether tumor-derived MIF leads to suppression of T cell immunity in vivo, we generated MIF-deficient neuroblastoma cell lines using short hairpin small interfering RNAs (siRNA). The MIF knockdown (MIFKD) AGN2a neuroblastoma cells were more effectively rejected in immune-competent mice than control siRNA-transduced or wild-type AGN2a. However, the increased rejection of MIFKD AGN2a was not observed in T cell-depleted mice. MIFKD tumors had increased infiltration of CD8(+) and CD4(+) T cells, as well as increased numbers of macrophages, dendritic cells, and B cells. Immunization with MIFKD AGN2a cells significantly increased protection against tumor challenge as compared with immunization with wild-type AGN2a, and the increased protection correlated with elevated frequencies of tumor-reactive CD8(+) T cells in the lymphoid tissue of treated animals. Increased numbers of infiltrating tumor-reactive CD8(+) T cells were also observed at the site of tumor vaccination. In vitro, treatment of AGN2a-derived culture supernatants with neutralizing MIF-specific Ab failed to reverse T cell suppressive activity, suggesting that MIF is not directly responsible for the immune suppression in vivo. This supports a model whereby MIF expression in neuroblastoma initiates a pathway that leads to the suppression of T cell immunity in vivo.     

Immediate transfection of patient-derived leukemia: a novel source for generating cell-based vaccines

Background

The production of cell-based cancer vaccines by gene vectors encoding proteins that stimulate the immune system has advanced rapidly in model systems. We sought to develop non-viral transfection methods that could transform patient tumor cells into cancer vaccines, paving the way for rapid production of autologous cell-based vaccines.

Methods

As the extended culture and expansion of most patient tumor cells is not possible, we sought to first evaluate a new technology that combines electroporation and chemical transfection in order to determine if plasmid-based gene vectors could be instantaneously delivered to the nucleus, and to determine if gene expression was possible in a cell-cycle independent manner. We tested cultured cell lines, a primary murine tumor, and primary human leukemia cells from diagnostic work-up for transgene expression, using both RFP and CD137L expression vectors.

Results

Combined electroporation-transfection directly delivered plasmid DNA to the nucleus of transfected cells, as demonstrated by confocal microscopy and real-time PCR analysis of isolated nuclei. Expression of protein from plasmid vectors could be detected as early as two hours post transfection. However, the kinetics of gene expression from plasmid-based vectors in tumor cell lines indicated that optimal gene expression was still dependent on cell division. We then tested to see if pediatric acute lymphocytic leukemia (ALL) would also display the rapid gene expression kinetics of tumor cells lines, determining gene expression 24 hours after transfection. Six of 12 specimens showed greater than 17% transgene expression, and all samples showed at least some transgene expression.

Conclusion

Given that transgene expression could be detected in a majority of primary tumor samples analyzed within hours, direct electroporation-based transfection of primary leukemia holds the potential to generate patient-specific cancer vaccines. Plasmid-based gene therapy represents a simple means to generate cell-based cancer vaccines and does not require the extensive infrastructure of a virus-based vector system.     

Breathing of awake goats during prolonged dysfunction of caudal M ventrolateral medullary neurons

Forster, H. V., L. G. Pan, T. F. Lowry, T. Feroah, W. M. Gershan, A. A. Whaley, M. M. Forster, and B. Sprtel. Breathing ofawake goats during prolonged dysfunction of caudal M ventrolateral medullary neurons. J. Appl. Physiol.84(1): 129-140, 1998.Cooling the caudal M ventrolateralmedullary (VLM) surface for 30 s results in a sustained apnea inanesthetized goats but only a 30% decrease in breathing in awakegoats. The purpose of the present study was to determine, in the awakestate, the effect of prolonged (minutes, hours) caudal M neuronaldysfunction on eupneic breathing andCO₂ sensitivity. Dysfunction wascreated by ejecting excitatory amino acid receptor antagonists or aneurotoxin on the VLM surface through guide tubes chronically implantedbilaterally on a 10- to 12-mm²portion of the caudal M VLM surface of 12 goats. Unilateral and bilateral ejections (1 µl) of selective antagonists forN-methyl-D-aspartic acid ornon-N-methyl-D-asparticacid receptors had no significant effect on eupneic breathing orCO₂ sensitivity. Unilateralejection of a nonselective excitatory amino acid receptor antagonistgenerally had no effect on eupneic breathing orCO₂ sensitivity. However, bilateral ejection of this antagonist resulted in a significant 2-Torrhypoventilation during eupnea and a significant reduction inCO₂ sensitivity to 60 ± 9% ofcontrol. Unilateral ejection of the neurotoxin kainic acid initiallystimulated breathing; however, breathing then returned to near controlwith no incidence of apnea. After the kainic acid ejection,CO₂ sensitivity was reducedsignificantly to 60 ± 7% of control. We conclude that in the awakestate a prolonged dysfunction of caudal M VLM neurons results incompensation by other mechanisms (e.g., carotid chemoreceptors, wakefulness) to maintain near-normal eupneic breathing, butcompensation is more limited for maintainingCO₂ sensitivity.

     

Mechanism of failure of recovery from hypoxic apnea by gasping in 17- to 23-day-old mice.

The mechanism of failure of autoresuscitation from hypoxic apnea in 17- to 23-day-old (weanling) Swiss Webster related/J mice was investigated by recording electrocardiogram (ECG) and ventilation in adult, weanling, and 11-day-old mice. Hypoxic apnea was induced with 97% N2-3% CO2. O2 (21% or 50% O2) or 97% N2-3% CO2 was given at the onset of apnea. The ECG showed no arrhythmias predictive of failure of autoresuscitation. The first indication of failure was a progressive fall in gasp volume ("run down"). This pattern also occurred in animals given continuous 97% N2-3% CO2 and was significantly different from that in mice that survived. Gasping duration in 97% N2 was longer in weanlings than adults but shorter than in 11 day olds. Respiratory and heart rate recovery were more rapid in adults than in weanlings. Although recovery in high O2 was more rapid, the survival rate was not increased. The lack of effect of high O2 on survival and the virtually identical pattern of gasping in mice dying in 97% N2 and air leads us to conclude that in mice that fail to autoresuscitate little or no O2 reaches the medullary respiratory centers. We speculate that this may be due to increased vulnerability of cardiac muscle to anoxia in 17- to 23-day-old mice, resulting in early and severe heart failure.