Regulation of B-CLL apoptosis through membrane receptors and Bcl-2 family proteins

The accumulation of monoclonal chronic lymphocytic leukemia B (B-CLL) cells may be due to excessive proliferation and longevity. Clinical progression may thus come from a constitutive but altered expression of a number of genes that results in extended B-CLL cells life span, increased proliferative capacity and diminished cell death. B-CLL cells express a number of surface markers that characterise the normal B-cells phenotype. However, B-CLL cells are CD5 positive and most of them also express CD6, surface receptors that are present in just a small subset of normal B-cells. When exploring CD6 function, we found out that cross-linking of CD6 protected B-CLL from anti-IgM-induced apoptosis. CD6 activation blocked anti-IgM- induced Bax(alpha) up-regulation and, by doing so, corrected an imbalance in the Bcl-2/Bax ratio that accompanies apoptosis. Here, we review all surface receptors and cytokines that have been described as participating in the induction or protection of B-CLL apoptosis together with data on chemosensitivity and gene modulation, data on the Fas receptor/Fas ligand system, and the implications of all the latter for B-CLL cell survival.     

Anti-AIDS agents. 33. Synthesis and anti-HIV activity of mono-methyl substituted 3',4'-di-O-(-)-camphanoyl-(+)-cis-khellactone (DCK) analogues

Four isomeric methyl substituted DCK analogues (2-5) were asymmetrically synthesized from different starting materials. 3-Methyl, 4-methyl, and 5-methyl-3',4'-di-O-(-)-camphanoyl-(+)-cis-khellactone (2-4) all were extremely potent against HIV-1 replication in H9 lymphocyte cells with EC50 and therapeutic index values of < 4.23 x 10(-7) microM and > 3.72 x 10(8), respectively, which are much better than those of DCK and AZT in this assay.     

CD4+ Th2 cells specific for mycobacterial 65-kilodalton heat shock protein protect against pristane-induced arthritis

Previous studies showed that mice with pristane-induced arthritis (PIA) and those protected from the disease by preimmunization with mycobacterial 65-kDa heat shock protein (hsp65), possess raised immune responses to hsp65. Thus, a paradox exists whereby T cells from both arthritic and hsp65-protected animals proliferate vigorously in response to the same Ag. Here we demonstrate that T cells from mice with PIA and hsp65-protected mice produce different cytokines in vitro in response to hsp65. The use of a sensitive CelELISA to measure Ag-driven lymphokine production revealed that spleen cells from hsp65-protected mice, but not those from pristane-injected or normal mice, produced the Th2-associated cytokines IL-4, IL-5, and IL-10 in response to stimulation with hsp65. By contrast, the Th1-associated cytokines IL-2 and IFN-gamma were produced by spleen cells from mice of all groups in response to hsp65. Furthermore, there was a dramatic increase in the IgG1 to IgG2a ratio of anti-hsp65 Abs from arthritic to protected mice. Thus, it appears that a Th2 response is protective against PIA. To examine this theory, a regimen of IL-12 administration which polarizes the hsp65-specific (Th2) immune response toward Th1 was identified. This regime abolished hsp65-mediated protection against PIA. Other experiments revealed that the specificity of the response to hsp65 was important, as other bacterial proteins known not to protect against PIA induced similar Th2-associated cytokines in vitro. It is considered that the protection afforded by hsp65 preimmunization is mediated by Th2-associated cytokines produced by hsp65-specific CD4+ T cells.     

Mutational analysis of the role of the distal histidine and glutamine residues of prostaglandin-endoperoxide synthase-2 in peroxidase catalysis, hydroperoxide reduction, and cyclooxygenase activation

Site-directed mutants of prostaglandin-endoperoxide synthase-2 (PGHS-2) with changes in the peroxidase active site were prepared by mutagenesis, expressed in Sf-9 cells, and purified to homogeneity. The distal histidine, His193, was mutated to alanine and the distal glutamine, Gln189, was changed to asparagine, valine, and arginine. The guaiacol peroxidase activities of H193A, Q189V, and Q189R were drastically reduced to levels observed in the absence of protein; only Q189N retained wild-type PGHS-2 (wtPGHS-2) activity. The mechanism of hydroperoxide reduction by the PGHS-2 mutants was investigated using 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a diagnostic probe of hydroperoxide reduction pathways. The hydroperoxide reduction activity of Q189V and Q189R was reduced to that of free Fe(III) protoporphyrin IX levels, whereas Q189N catalyzed more reduction events than wtPGHS-2. The percentage of two-electron reduction events was identical for wtPGHS-2 and Q189N. The number of hydroperoxide reductions catalyzed by H193A was reduced to approximately 60% of wtPGHS-2 activity, but the majority of products were the one-electron reduction products, 15-KETE and epoxyalcohols. Thus, mutation of the distal histidine to alanine leads to a change in the mechanism of hydroperoxide reduction. Reaction of wtPGHS-2, Q189N, and H193A with varying concentrations of 15-HPETE revealed a change in product profile that suggests that 15-HPETE can compete with the reducing substrate for oxidation by the peroxidase higher oxidation state, compound I. The ability of the PGHS-2 proteins to catalyze two-electron hydroperoxide reduction correlated with the activation of cyclooxygenase activity. The reduced ability of H193A to catalyze two-electron hydroperoxide reduction resulted in a substantial lag phase in the cyclooxygenase assay. The addition of 2-methylimidazole chemically reconstituted the two-electron hydroperoxide reduction activity of H193A and abolished the cyclooxygenase lag phase. These observations are consistent with the involvement of the two-electron oxidized peroxidase intermediate, compound I, as the mediator of the activation of the cyclooxygenase of PGHS.     

Adrenal androgen excess in the polycystic ovary syndrome: sensitivity and responsivity of the hypothalamic-pituitary-adrenal axis

Over 50% of patients with the polycystic ovary syndrome (PCOS) demonstrate excess levels of adrenal androgens (AAs), particularly dehydroepiandrosterone sulfate (DHS). Nonetheless, the mechanism for the AA excess remains unclear. It has been noted that in PCOS the pituitary and ovarian responses to their respective trophic factors (i.e. GnRH and LH, respectively) are exaggerated. Similarly, we have postulated that excess AAs in PCOS arises from dysfunction of the hypothalamic-pituitary-adrenal axis, due to 1) exaggerated pituitary secretion of ACTH in response to hypothalamic CRH, 2) excess sensitivity/responsivity of AAs to ACTH stimulation, or 3) both. To test this hypothesis we studied 12 PCOS patients with AA excess (HI-DHS; DHS, > 8.1 mumol/L or 3000 ng/mL), 12 PCOS patients without AA excess (LO-DHS; DHS, < 7.5 mumol/L or 2750 ng/mL), and 11 controls (normal subjects). Each subject underwent an acute 90-min ovine CRH stimulation test (1 microgram/kg) and an 8-h incremental i.v. stimulation with ACTH-(1-24) at doses ranging from 20-2880 ng/1.5 m2.h) with a final bolus of 0.25 mg. All patient groups had similar mean body mass indexes and ages, and both tests were performed in the morning during the follicular phase (days 3-10) of the same menstrual cycle, separated by 48-96 h. During the acute ovine CRH stimulation test, no significant differences in the net maximal response (i.e. change from baseline to peak level) for ACTH, dehydroepiandrosterone (DHA), androstenedione (A4), or cortisol (F) or for the DHA/ACTH, A4/ACTH, or F/ACTH ratios was observed. Nonetheless, the net response of DHA/F and the areas under the curve (AUCs) for DHA and DHA/F indicated a greater response for HI-DHS vs. LO-DHS or normal subjects. The AUC for A4 and A4/F and the delta A4/delta F ratio (delta = net maximum change) indicated that HI-DHS and LO-DHS had similar responses, which were greater than that of the normal subjects, although the difference between LO-DHS patients and normal subjects reached significance only for the AUC of the A4 response. No difference in the sensitivity (i.e. threshold or minimal stimulatory dose) to ACTH was noted between the groups for any of the steroids measured. Nonetheless, the average dose of ACTH-(1-24) required for a threshold response was higher for DHA than for F and A4 in all groups. No difference in mean responsivity (slope of response to incremental ACTH stimulation) was observed for DHA and F between study groups, whereas the responsivity of A4 was higher in HI-DHS patients than in normal or LO-DHS women. The net maximal and the overall (i.e. AUC) responses of DHA were greater for HI-DHS than for normal or LO-DHS women. The response of A4 and the delta A4/delta F ratio were greater for HI-DHS patients than for LO-DHS patients or normal subjects. Alternatively, HI-DHS and LO-DHS patients had similar overall responses (i.e. AUC) for A4 or A4/F, although both were greater than those of normal subjects. The relative differences in response to incremental ACTH stimulation between steroids was consistent for all subject groups studied, i.e. A4 > F or DHA. In conclusion, our data suggest that AA excess in PCOS patients is related to an exaggerated secretory response of the adrenal cortex for DHA and A4, but not to an altered pituitary responsivity to CRH or to increased sensitivity of these AAs to ACTH stimulation. Whether the increased responsivity to ACTH for these steroids is secondary to increased zonae reticularis mass or to differences in P450c17 alpha activity, particularly of the delta 4 pathway, remains to be determined.     

Solitary scapula mass: atypical presentation of esophageal adenocarcinoma

The stability of adenosine in various diluents in polypropylene syringes and polyvinyl chloride (PVC) bags at three temperatures was studied. Portions of pooled undiluted adenosine infusion (3 mg/ mL) were stored in 60-mL capped syringes, 20 for each storage condition. Adenosine infusions were prepared by mixing adenosine with 5% dextrose injection, 0.9% sodium chloride injection, lactated Ringer's injection, or 5% dextrose and lactated Ringer's injection to produce a concentration of 0.75 mg/mL. Samples of each infusion were stored in 60-mL capped syringes and 50-mL bags, 20 syringes and 20 bags for each storage condition. Syringes and bags were stored in the dark at 25, 5, and -15 degrees C. At various sampling times, three syringes and three bags of each infusion were removed for visual inspection, pH measurement, and high-performance liquid chromatographic analysis. At 10 and 16 days, fungal growth at 25 degrees C was suspected in the infusions prepared with 5% dextrose injection. For all other samples, there was no evidence of precipitation or change in pH. The concentration of adenosine remained constant in all samples at all storage conditions. Adenosine 3 mg/mL was stable in polypropylene syringes for 7 days at 25 degrees C, 14 days at 5 degrees C, and 28 days at -15 degrees C; adenosine 0.75 mg/ mL in 0.9% sodium chloride injection and in 5% dextrose injection was stable in polypropylene syringes and PVC bags for 16 days at 25, 5, and -15 degrees C; and adenosine 0.75 mg/mL in lactated Ringer's injection and in 5% dextrose and lactated Ringer's injection was stable in syringes and bags for 14 days at 25, 5, and -15 degrees C.     

Antibiotic properties of bovine lactoferrin on Helicobacter pylori

To investigate a potential new treatment for gastric Helicobacter pylori infection, we have examined the use of the natural antibiotic lactoferrin, found in bovine milk, for activity against Helicobacter species both in vitro and in vivo. Lactoferrin was bacteriostatic to H. pylori when cultured at concentrations > or =0.5 mg/ml. Growth of H. pylori was not inhibited by another milk constituent, lysozyme, or by a metabolite of lactoferrin, lactoferricin B, but growth was inhibited by the iron chelator deferoxamine mesylate. Lactoferrin inhibition of growth could be reversed by addition of excess iron to the medium. Lactoferrin in retail dairy milk was found to be more stable intragastrically than unbuffered, purified lactoferrin. Treatment of H. felis-infected mice with lactoferrin partially reversed mucosal disease manifestations. It is concluded that bovine lactoferrin has significant antimicrobial activity against Helicobacter species in vitro and in vivo. Bovine lactoferrin should be further investigated for possible use in H. pylori infections in man.