DNA aptamers block L-selectin function in vivo. Inhibition of human lymphocyte trafficking in SCID mice.

Selectins participate in the initial events leading to leukocyte extravasation from the blood into tissues. Thus the selectins have generated much interest as targets for antiinflammatory agents. Therapeutic molecules based on the monomeric carbohydrate ligand sialyl Lewis X (SLe(X)) have low affinities and are not specific for a given selectin. Using SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technology, we have generated aptamers specific for L-selectin that require divalent cations for binding and have low nanomolar affinity. In vitro, the deoxyoligonucleotides inhibit L-selectin binding to immobilized SLe(X) in static assays and inhibit L-selectin-mediated rolling of human lymphocytes and neutrophils on cytokine-activated endothelial cells in flow-based assays. These aptamers also block L-selectin-dependent lymphocyte trafficking in vivo, indicating their potential utility as therapeutics.     

Mutant p53 exerts a dominant negative effect by preventing wild-type p53 from binding to the promoter of its target genes

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer. A majority of these mutations are missense mutations in the DNA-binding domain. As a result, the mutated p53 gene encodes a full-length protein incapable of transactivating its target genes. In addition to this loss of function, mutant p53 can have a dominant negative effect over wild-type p53 and/or gain of function activity independently of the wild-type protein. To better understand the nature of the tumorigenic activity of mutant p53, we have investigated the mechanism by which mutant p53 can exert a dominant negative effect. We have established several stable cell lines capable of inducibly expressing a p53 mutant alone, wild-type p53 alone, or both proteins concurrently. In this context, we have used chromatin immunoprecipitation to determine the ability of wild-type p53 to bind to its endogenous target genes in the presence of various p53 mutants. We have found that p53 missense mutants markedly reduce the binding of wild-type p53 to the p53 responsive element in the target genes of p21, MDM2, and PIG3. These findings correlate with the reduced ability of wild-type p53 in inducing these and other endogenous target genes and growth suppression in the presence of mutant p53. We also showed that mutant p53 suppresses the ability of wild-type p53 in inducing cell cycle arrest. This highlights the sensitivity and utility of the dual inducible expression system because in previous studies, p53-mediated cell cycle arrest is not affected by transiently overexpressed p53 mutants. Together, our data showed that mutant p53 exerts its dominant negative activity by abrogating the DNA binding, and subsequently the growth suppression, functions of wild-type p53.     

Integration of family planning into maternal and child health and other services at Harare Central Hospital

To increase the accessibility, availability, and acceptance of family planning methods and counseling, family planning services were integrated with maternal and child health care services at Harare Central Hospital in Zimbabwe. The Family Planning Project was implemented with hopes that mothers would seek contraceptive methods and counseling concurrent with their or their children's hospital admission, thereby making facility and service inaccessibility a thing of the past. Ante-natal, post-natal, and postabortal women were targeted for project outreach at the facility, along with patients suffering chronic medical and psychiatric problems, and mothers of malnourished children. Weaknesses of family planning provision at the hospital prior to the project are presented in the component parts of pharmacy, emergency gynecology unit, outpatient department, ante-natal clinic, post-partum care, post-natal clinic, and the general hospital. 2 full-time nurse- midwives and 2 part-time gynecologists counsel and provide services for the Family Planning Project. Other programmatic changes and improvements are described. There were 3,822 new acceptors and 5,423 return visits during the 1st project year, with the nurse-midwives providing 3,114 couple-years of protection, equal to 5.1% of the total provided by all 35 national family planning council clinics. Additional results, plans for the future, and problem areas are further discussed. The project, undertaken with few resources and high motivation, yielded high family planning acceptance rates with markedly less inconvenience for acceptors.     

Comparison of the radiographic vertebral trabecular pattern with the vertebral fracture prevalence and spinal bone density

Spinal bone densitometry allows accurate and precise measurement of the severity of bone loss. Where densitometry is not yet available medical practitioners have to continue to rely on clinical radiography. Since the grey levels of the radiographic image are highly inaccurate we studied the radiographic vertebral trabecular pattern for its suitability as a semiquantitative assessment of vertebral bone loss. We defined four vertebral trabecular pattern indices (VTPI 4=normal, VTPI 1=severe bone loss) and tested these for correlations with the prevalence of vertebral fractures, and with spinal and hip bone mineral density measured by dual-energy X-ray absorptiometry (DXA). We found negative correlations between VTPI and the percentage of patients with vertebral fractures (p=0.0001), between VTPI and the number of vertebral fractures per patient (r=0.606,p=0.001) and between VTPI and the severity of vertebral fractures, and a positive correlation between VTPI and spinal (r ²=0.556,p=0.0001) and hip DXA values (r ²=0.315,p=0.0001). We conclude that the vertebral trabecular pattern index can be used to assess the severity of spinal bone loss when a bone densitometer is not available.     

Rapid amplification of immunoglobulin heavy chain switch (IGHS) translocation breakpoints using long-distance inverse PCR.

Molecular cloning of immunoglobulin heavy chain (IGH) translocation breakpoints identifies genes of biological importance in the development of normal and malignant B cells. Long-distance inverse PCR (LDI-PCR) was first applied to amplification of IGH gene translocations targeted to the joining (IGHJ) regions. We report here successful amplification of the breakpoint of IGH translocations targeted to switch (IGHS) regions by LDI-PCR. To detect IGHS translocations, Southern blot assays using 5' and 3' switch probes were performed. Illegitimate Smu rearrangements were amplified from the 5' end (5'Smu LDI-PCR) from the alternative derivative chromosome, and those of Sgamma or Salpha were amplified from the 3' end (3'Sgamma or 3'alpha LDI-PCR) from the derivative chromosome 14. Using a combination of these methods, we have succeeded in amplifying IGHS translocation breakpoints involving FGFR3/MMSET on 4p16, BCL6 on 3q27, MYC on 8q24, IRTA1 on 1q21 and PAX5 on 9p13 as well as BCL11A on 2p13 and CCND3 on 6p21. The combination of LDI-PCR for IGHJ and IGHS allows rapid molecular cloning of almost all IGH gene translocation breakpoints.     

Functional residual capacity and lung mechanics at different levels of mechanical ventilation

We assessed the effects of rapid ventilatory rates (60 to 120 breath/min) and high mechanical ventilation pressures (30/5 to 40/10 cm H2O) on lung mechanics and intravascular pressures in 9 paralyzed, sedated rabbits ventilated with a time-cycled, pressure-limited flow generator (Baby bird). Measurements of tidal volume, ventilator line pressure, tracheal pressure, functional residual capacity (FRC), and arterial and venous blood pressures showed that: 68% of the peak pressure developed by the ventilator was transmitted to the trachea at 60 breath/min, 74% at 120 breath/min, and 87% when ventilation pressures were increased to 40/10 cm H2O; when the ventilatory rate and the PEEP were increased, the end-expiratory pressure in the trachea became progressively greater than that indicated on the ventilator pressure gauge; FRC increased when the PEEP and mean tracheal pressure increased; tidal volume and dynamic compliance decreased and minute ventilation increased as ventilatory rate increased; compliance decreased whenever FRC increased, and increased whenever FRC decreased; and there was little effect on mean central venous or arterial pressure. These data indicate that increasing ventilator rates cause gas trapping within the lung. In normal animals, this may interfere with gas exchange and pulmonary blood flow. In abnormal lungs, the gas trapping may increase FRC and improve gas exchange within the lung.     

Hormonal imbalance and alterations in testicular morphology induced by chronic ingestion of ethanol

Physical correlates of chronic ethanol consumption and measures of in vitro fertilization were examined in male C57B1 mice after a treatment period of 34 days with a total liquid nutriment diet which contained 5%/95% (v/v) ethanol. This diet represented relatively low levels of ethanol ingestion by the C57B1 mouse, as evidenced by the low peak levels of blood ethanol (160 mg/100 ml), the lack of behavioral signs of intoxication during treatment, and the absence of overt signs of physical dependence after withdrawal from the ethanol-containing diet. Plasma testosterone levels of experimental animals were depressed during the treatment period, as compared to testosterone levels of pair-fed controls. No evidence of hepatic injury was observed following the treatment period. Although fertility, as measured by the ability of spermatozoa to penetrate mouse ova in vitro, was unaffected by the chronic ethanol treatment, signs of testicular dysfunction were evident. Abnormal testicular morphology included disruption of the basement membranes of the seminiferous tubules, decreased tubular diameter, and desquamation of immature germ cells into the lumina of the tubules. The present study provides convincing evidence of the adverse effects upon male reproductive functions of relatively low levels of ethanol, when administered chronically, under controlled conditions.     

Orbitofrontal cortex lesions result in abnormal social judgements to emotional faces

Facial expressions of emotion display a wealth of important social information that we use to guide our social judgements. The aim of the current study was to investigate whether patients with orbitofrontal cortex (OFC) lesions exhibit an impaired ability to judge the approachability of emotional faces. Furthermore, we also intended to establish whether impaired approachability judgements provided to emotional faces emerged in the presence of preserved explicit facial expression recognition. Using non-parametric statistics, we found that patients with OFC lesions had a particular difficulty using negative facial expressions to guide approachability judgements, compared to healthy controls and patients with frontal lesions sparing the OFC. Importantly, this deficit arose in the absence of an explicit facial expression recognition deficit. In our sample of healthy controls, we also demonstrated that the capacity to recognise facial expressions was not significantly correlated with approachability judgements given to emotional faces. These results demonstrate that the integrity of the OFC is critical for the appropriate assessment of approachability from negatively valenced faces and this ability is functionally dissociable from the capacity to explicitly recognise facial expressions.