Aged-rodent models of long-term growth hormone therapy: lack of deleterious effect on longevity

Studies were carried out to examine the effects of long-term recombinant human growth hormone (GH) therapy on longevity in rodents. In the first study, 150 18-month-old female F344 rats were divided into three groups of 50 rats per group: Group 1, solvent vehicle; Group 2, 10 microg GH/kg body weight three times per week; Group 3, 50 microg GH/kg body weight three times per week. GH and solvent vehicle therapies were started at 18 months of age and continued until all the animals died spontaneously. Serum insulin-like growth factor (IGF)-I was measured at 18 and 29 months of age and on 3-month-old rats. Serum IGF-I level decreased between 3 and 29 months of age. GH therapy reversed the decrease in a dose-dependent manner, with the 50 microg GH dose returning the serum IGF-I level to that of 3-month-old animals. However, statistical analysis revealed no significant effect of GH therapy on median life span, 10th percentile life span, or maximum life span. Similar observations on longevity were made on aged F344 male rats and on aged Balb/c mice, even when the dose of GH was increased to 1.0 mg/kg body weight two times per week. The main pathologic lesions in control animals were nephropathy, cardiomyopathy, leukemia, and testicular interstitial cell tumor; the prevalence of these lesions was not significantly altered by GH therapy. We conclude that long-term low-dose GH therapy that includes doses in the range that is given to humans in clinical trials in GH deficiency and to revert age-related physiologic declines has no overt deleterious effects on longevity and pathology in aged rodents.     

Pharmacological characterization of the pseudopterosins: novel anti-inflammatory natural products isolated from the Caribbean soft coral, Pseudopterogorgia elisabethae

Pseudopterosin E (PSE), a C-10 linked fucose glycoside and pseudopterosin A (PSA), a C-9 xylose glycoside isolated from the marine gorgonian Pseudopterogorgia elisabethae were both effective in reducing PMA-induced mouse ear edema when administered topically (ED50 (microg/ear) PSE(38), PSA(8)) or systemically (ED50 (mg/kg, i.p.) PSE (14), PSA (32)). Both compounds exhibited in vivo analgesic activity in phenyl-p-benzoquinone-induced writhing (ED50 (mg/kg, i.p.) PSE(14), PSA(4). PSE inhibited zymosan-induced writhing (ED50 = 6 mg/kg, i.p.), with a concomitant dose-dependent inhibition of peritoneal exudate 6-keto-prostaglandin F1alpha (ED50 = 24 mg/kg) and leukotriene C4 (ED50 = 24 mg/kg). In vitro, the pseudopterosins were inactive as inhibitors of phospholipase A2, cyclooxygenase, cytokine release, or as regulators of adhesion molecule expression. PSA inhibited prostaglandin E2 and leukotriene C4 production in zymosan-stimulated murine peritoneal macrophages (IC50 = 4 microM and 1 microM, respectively); however, PSE was much less effective. These data suggest that the pseudopterosins may mediate their anti-inflammatory effects by inhibiting eicosanoid release from inflammatory cells in a concentration and dose-dependent manner.     

Renal arteriovenous fistulas

Sinusoidal variations of CO2 were applied to anesthetized White Leghorn cockerels enclosed in a body plethysmograph. Waveforms were 2.5% mean plus 2.5% peak sine wave CO2 in air, at frequencies of 0.011 to 0.467 rad/s. Reflex variation in tidal volume and respiratory interval in response to the inputs of sinusoidally varying fractional content of inhaled CO2 for nine points within the above range of driving frequencies were recorded breath-by-breath. A least squares best-fit procedure was used to estimate mathematical input-output relationships (transfer functions) between the outputs, tidal volume (VT) and respiratory interval (1/f), and the single input, inspired CO2 fraction. For VT, a three time constant model most closely fitted the data. Two time constants, 80 s and 3.9 s, were associated with negligible delay; the third, 14 s, has a 0.35 s time delay. Dynamics of VT and interval reflex responses differed significantly suggesting that they are regulated by separate mechanisms.     

The resolution of membrane proteins based upon size, charge, and hydrophobicity

Currently available systems for resolving membrane proteins are based only on size and charge differences. Recently, it has been shown that Triton-urea-acetic acid gels which separate proteins on the basis of charge, size and hydrophobicity are capable of resolving proteins differing only by the substitution of a single neutral amino acid. We have applied this new method to the resolution of bacterial envelope proteins. Conditions for optimal resolution of different bacterial envelope proteins were determined by electrophoresis through transverse urea and Triton X-100 gradient gels. We have also correlated the components resolved in this system with those resolved by classical sodium dodecyl sulfate-gel electrophoresis by using two-dimensional slab gels combining the two systems. Furthermore, envelope protein fractions from different species and strains of bacteria were compared to identify specific proteins. This system appears to be a promising method for investigating envelope proteins which are due to missense mutations.     

Expression of mRNAs encoding subunits of the N-methyl-D-aspartate receptor in cultured cortical neurons

The expression of mRNAs encoding subunits of the N-methyl-D-aspartate (NMDA) receptor was examined in cortical neurons maintained in primary culture. Cultures were prepared from embryonic day 17 rat neocortex. At this developmental age, levels of NR1, NR2A, NR2B, and NR2C mRNA were low or undetectable. Expression of NR1 mRNA increased progressively between days 1 and 21 in vitro. The amount of NR2A mRNA did not change between days 1 and 7 but increased between days 7 and 21. In contrast, levels of NR2B mRNA increased between days 1 and 7, with little further change after day 7. The level of NR2B mRNA was approximately 4-fold higher than that of NR2A mRNA in 21-day cultures. Using ligand binding assays, the proportion of NMDA receptors having a low affinity for ifenprodil was also found to increase over time in culture. The increase in the expression of receptors having a low affinity for ifenprodil and the increase in NR1 and NR2A mRNAs were reduced or prevented by maintaining cells in medium with a low concentration of serum. The results are consistent with the hypothesis that inclusion of the NR2A subunit in native NMDA receptors is responsible for their low affinity for ifenprodil. Splice variants of NR1 lacking the 5' (amino-terminal) insert were found to be the predominant forms of NR1 in cultured neurons. Variants containing the 5' insert represented only a small (< or = 5%) fraction of total NR1 mRNA, and their proportion was not altered as a function of time in culture. Time-dependent changes in the properties of NMDA receptors and in the expression of subunit mRNA occurring in cultured neurons are similar to changes observed in developing rat brain. Thus, the developmental sequence of NMDA receptor expression that occurs in vivo is partially retained in neurons maintained in vitro.     

Comparing functional results one year and ten years after ileal pouch-anal anastomosis for chronic ulcerative colitis

Proctocolectomy with ileal pouch-anal anastomosis (IPAA) is the treatment of choice for most patients with chronic ulcerative colitis. Long-term results, however remain undefined; the major concern is that function may deteriorate. PURPOSE: The aim of this study was to assess functional outcome in a subgroup of patients who have an IPAA for chronic ulcerative colitis for > 10 years. METHODS: Among 1400 IPAA patients, 75 consecutive subjects (31 females and 44 males; median age 31 at operation) were identified who had the procedure prior to 1982. All patients had functional results recorded 1 year and 10 years following ileostomy closure. RESULTS: There were four deaths during the follow-up period; none were pouch related. Two patients refused ileostomy closure. Of the remaining 69 patients, there were 8 (11 percent) failures, leaving 61 subjects available for study. Stool frequency (7 +/- 3, mean +/- SD) remained unchanged. Of the 50 subjects with initially excellent daytime continence, 39 (78 percent) remained the same, 10 (20 percent) developed minor incontinence, and 1 developed poor control after 10 years. Four of 10 subjects (40 percent) with initial minor daytime incontinence remained unchanged, 4 (40 percent) improved, and 2 (20 percent) worsened. The one subject with poor control at one year was unchanged. Nocturnal fecal spotting increased over the 10-year period but not significantly (38 percent vs. 52 percent; P = 0.08). CONCLUSIONS: After IPAA, functional results in terms of stool frequency and rate of fecal incontinence did not deteriorate with time.     

CD4-mediated and CD8-mediated cytotoxic and proliferative immune responses to Toxoplasma gondii in seropositive humans

Both CD4+ and CD8+ cytotoxic T lymphocytes (CTL) are part of the human immune response to Toxoplasma gondii infection. To further our understanding of Toxoplasma immunity, we investigated factors influencing stimulation of CD4+ or CD8+ human T. gondii-specific immune cells. Both antigen-pulsed and Toxoplasma-infected antigen-presenting cells (APC) induced cell proliferation. Toxoplasma-infected APC elicited strong proliferation of CD4+ cells, but little or no proliferation of CD8+ cells, unless high antigen loads were used. Toxoplasma-infected APC stimulated specific cytotoxicity poorly or not at all, owing to death of stimulated cultures, whereas antigen-pulsed APC strongly elicited specific cytotoxicity. Cytotoxicity elicited by either type of APC resided exclusively in CD4+ T cells in polyclonal cultures. Thus, Toxoplasma-infected APC elicited stronger CD4-mediated than CD8-mediated cell proliferation and generated CD4+ CTL more readily than CD8+ CTL. Nonetheless, specific CD8+ memory cells were demonstrated, and rare CD8+ Toxoplasma-specific CTL were subcloned. Fixed Toxoplasma-infected APC (which induce CD8+ CTL) also elicited cell proliferation, but polyclonal cultures stimulated with these infected APC did not die. Unfixed Toxoplasma-infected APC strongly inhibited phytohemagglutinin-induced cell proliferation, whereas fixed APC did not. These data suggested that infected APC were inhibitory or lethal to some immune cells. Further investigations into interactions between immune cells and Toxoplasma-infected cells likely will help elucidate factors involved in the immunopathogenesis of Toxoplasma infection. As other intracellular parasites, including Plasmodium spp. and Leishmania spp., also elicit CD4+ CTL, such work may help establish paradigms governing immunity to intracellular parasites.