Generation of CFU-C suppressor T cells in vitro. III. Failure of mitogen-primed T cells from patients with chronic granulocytic leukemia to inhibit the growth of normal CFU-C

T lymphocytes were derived by E rosetting from the peripheral blood (PB) and bone marrow (BM) of 15 patients with chronic granulocytic leukemia (CGL) in the chronic phase of their disease. T cells were also obtained from 12 healthy individuals. T cells were incubated overnight either in culture medium (RPMI) or RPMI plus pokeweed mitogen (PWM). The supernatants were then recovered and the cells washed in fresh RPMI. T cells from normal donors and from CGL patients were then cocultured with normal allogeneic marrow cells grown in soft agar for CFU-C colony formation. Target marrow cells were also grown in agar in the presence of T-derived supernatants. The results of this study can be summarized as follows. (1) Normal PB and BM T cells efficiently suppressed autologous and allogeneic CFU-C growth after PWM stimulation. (2) T cells derived from peripheral blood or marrow of CGL patients failed to inhibit CFU-C growth, whether pretreated with PWM or not. (3) The supernatants of PWM-treated normal T cells strongly inhibited CFU-C colony formation, whereas the supernatants of PWM- treated CGL T cells had no CFU-C/suppressor activity. These data indicate that T cells from CGL patients cannot be primed to become CFU- C suppressor cells after PWM: stimulation in vitro and cannot release a soluble inhibitor of granulopoiesis produced by PWM-primed normal T cells.     

Modified antisense oligodeoxynucleotides against the splice acceptor site of tat do not inhibit in vitro hematopoietic colony growth in HIV-positive patients

The hematopoietic failure in the majority of patients with progressive HIV infection is further aggravated by virustatic agents like azidothymidine. As an alternative therapeutic attempt, three derivatives of an antisense oligodeoxynucleotide (ODN) against the splice acceptor site of the tat gene have been shown to inhibit HIV replication in vitro. This study was aimed at examining whether these agents are toxic to the hematopoietic progenitor cells. To this end, bone marrow cells from HIV-positive and healthy persons were depleted from adherent cells to eliminate fibroblasts. In further experiments, the cells were additionally enriched for CD34-positive hematopoietic progenitor cells or were depleted fromTCS-1-positive T lymphocytes. At concentrations of 1.25–10 M, the three antisense ODN did not inhibit any erythrocyte or granulocyte-monocyte colony growth from CD34-positive cells, either from the HIV-positive or from the HIV-negative cohort. In contrast to azidothymidine, which served as inhibitory control, a significant increase of colony growth was seen after depletion of fibroblasts, ofTCS-1-positive cells, or without cell separation. In conclusion, the three oligodeoxynucleotides do not exert any hematotoxic effect but do increase colony formation from low-density bone marrow cells in vitro and could therefore be useful in future clinical studies.     

Focal segmental glomerulosclerosis in patients receiving lithium carbonate

Lithium carbonate is a commonly used psychiatric medication with a number of toxic renal effects, which include nephrotic-range proteinuria. A review of the literature concerning lithium-induced proteinuria is presented and three cases of nephrotic-range proteinuria are described in association with lithium therapy. The pathology in these three cases was focal segmental glomerulosclerosis, a finding not previously described.     

Physiological concentrations of tissue factor pathway inhibitor do not inhibit prothrombinase

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine proteinase inhibitor that directly inhibits factor Xa and, in a factor Xa dependent manner, inhibits the factor VIIa/tissue factor catalytic complex. The inhibitory effect of TFPI in prothrombin activation assays using purified components of the prothrombinase complex was examined. When factor Xa is added to mixtures containing TFPI, prothrombin, calcium ions, and nonactivated platelets or factor V and phospholipids, TFPI significantly reduces subsequent thrombin generation, and the inhibitory effect is enhanced by heparin. If factor Xa is preincubated with calcium ions and thrombin-activated platelets or factor Va and phospholipids to permit formation of prothrombinase before the addition of prothrombin and physiologic concentrations of TFPI (< 8 nmol/L), minimal inhibition of thrombin generation occurs, even in the presence of heparin. Thus, contrary to results in amidolytic assays with chromogenic substrates, prothrombinase is resistant to inhibition by TFPI in the presence of its physiological substrate, prothrombin. Higher concentrations of TFPI (approximately 100 nmol/L), similar to those used in animal studies testing for therapeutic actions of TFPI, do effectively block prothrombinase activity.     

Dabigatran and rivaroxaban do not affect AA- and ADP-induced platelet aggregation in patients receiving concomitant platelet inhibitors

Dabigatran and rivaroxaban are novel, vitamin K-independent oral anticoagulants (NOACs) and act via antagonism of the coagulation factor (F) IIa (dabigatran) or FXa (rivaroxaban), respectively. Compared to vitamin-K-antagonists, NOACs have shown non-inferiority of risk and benefit in patients with non valvular atrial fibrillation (AF). In clinical practice there is increasing use of NOACs combined with platelet inhibitors in patients with AF and coronary artery disease. However, whether NOACs affect the function of platelet inhibitors remains incompletely known. This observational study aimed to assess the platelet function in patients receiving dabigatran or rivaroxaban and concomitant platelet inhibitors. A single centre observational study was performed analysing the platelet aggregation of patients treated with dabigatran or rivaroxaban with or without concomitant platelet inhibitors. Measurements before the initiation of NOAC therapy served as the respective control group. Platelet aggregation was measured by multiple electrode aggregometry and was induced with adenosine diphosphate (ADP, 6.5 µM) and arachidonic acid (AA, 0.5 mM), respectively. In order to evaluate whether NOACs interact with platelet inhibition by ASA or the P2Y₁₂-antagonist clopidogrel, 87 patients were grouped according to their concomitant antiplatelet medication. Comparing the ADP- and AA-induced platelet aggregation in patients without concomitant platelet inhibitors (n = 45) no significant differences under therapy with dabigatran (d) or rivaroxaban (r) compared to the control group (c) were observed. In patients taking clopidogrel as a concomitant platelet inhibitor (n = 21), neither dabigatran nor rivaroxaban affected the ADP-induced platelet aggregation (c 20 ± 11, d 21 ± 14, r 18 ± 8 AU*min, p = 0.200). Patients receiving dabigatran or rivaroxaban in combination with ASA (n = 42; 21 ASA only, 21 ASA + clopidogrel) showed no significant differences of the AA-induced aggregation compared to the control group (c 10 ± 8, d 9 ± 7, r 10 ± 8 AU*min, p = 0.810). The antiplatelet effects of ASA and clopidogrel monitored by AA- or ADP-induced platelet aggregation were not affected by NOAC therapy.     

Release of growth promotors from mononuclear human blood cells by LPS, lectins and lithium, as tested in the mouse CFU-C assay

Medium (MCM, 20% human serum), conditioned for 24 h by mononuclear human blood cells, had no colony-forming ability when tested in the mouse CFU-C assay. However, when combined with L-CSF, which predominantly generates macrophage colonies, MCM increased the colony number and size. Even more significant was the increased cellularity with a striking shift towards granulocyte production. Some human sera induced GIF formation without additives, but mostly LPS was required. After heat inactivation, all sera became dependent on LPS to yield an active MCM. Lithium, PHA, Con A and PMW also stimulated GIF formation, which itself depended upon protein synthesis. Heat inactivation of LPS and serum did not reduce their ability, in combination, to yield an active MCM. However, when serum and LPS were mixed before heat treatment, the ability to induce GIF production was abolished, but could be restored by adding intact LPS. This may indicate that LPS exerts its effect by combining with a serum factor yielding a heat-sensitive complex. However, even in the absence of serum, LPS had a stimulatory effect when used in large concentrations, but still the MCM was less active than MCM with serum.     

Cobimetinib and trametinib inhibit platelet MEK but do not cause platelet dysfunction

The MEK inhibitors cobimetinib and trametinib are used in combination with BRAF inhibitors to treat metastatic melanoma but increase rates of hemorrhage relative to BRAF inhibitors alone. Platelets express several members of the MAPK signalling cascade including MEK1 and MEK2 and ERK1 and ERK2 but their role in platelet function and haemostasis is ambiguous as previous reports have been contradictory. It is therefore unclear if MEK inhibitors might be causing platelet dysfunction and contributing to increased hemorrhage. In the present study we performed pharmacological characterisation of cobimetinib and trametinib in vitro to investigate potential for MEK inhibitors to cause platelet dysfunction.

We report that whilst both cobimetinib and trametinib are potent inhibitors of platelet MEK activity, treatment with trametinib did not alter platelet function. Treatment with cobimetinib results in inhibition of platelet aggregation, integrin activation, alpha-granule secretion and adhesion but only at suprapharmacological concentrations. We identified that the inhibitory effects of high concentrations of cobimetinib are associated with off-target inhibition on Akt and PKC. Neither inhibitor caused any alteration in thrombus formation on collagen under flow conditions in vitro.

Our findings demonstrate that platelets are able to function normally when MEK activity is fully inhibited, indicating MEK activity is dispensable for normal platelet function. We conclude that the MEK inhibitors cobimetinib and trametinib do not induce platelet dysfunction and are therefore unlikely to contribute to increased incidence of bleeding reported during MEK inhibitor therapy.     

What an endocrinologist should know for patients receiving lithium therapy

Lithium is an efficient treatment of bipolar disorder. Besides renal insufficiency, many endocrine side effects are described such as the occurrence of thyroid disorders, hypercalcaemia and nephrogenic diabetes insipidus. Lithium inhibits the secretion of thyroid hormones. The prevalence of goiter is 4 times more common in Lithium-treated patients compared as to the general population. Hypothyroidism (8–20%) is more frequent in women and in case of pre-existing thyroid autoimmunity. Grave's disease and other hyperthyroidisms are sometimes reported. Lithium stimulates the proliferation of parathyroid cells by activating the Wnt pathway. An increase in serum calcium and PTH is described in patients treated with Lithium with a 4 to 6-fold higher risk of primary hyperparathyroidism than in the general population. Nevertheless, 24-hour urine calcium is not often increased, and the phenotype can mimic a hypercalcemia-hypocalciuria syndrome that may regress with Lithium discontinuation. Surgery should be cautious since parathyroid hyperplasia is more common than parathyroid adenoma. Nephrogenic diabetes insipidus is frequently reported and may be debilitating, sometimes intricated with severe dehydration, hypernatremia, and acute renal insufficiency. Nephrogenic diabetes insipidus is not generally reversible after Lithium discontinuation, especially in patients who have chronic kidney disease due to interstitial tubule nephritis. In conclusion, clinical assessment (goiter, diuresis) and biological monitoring of serum calcium, sodium creatinine, TSH and lithium are recommended in patients receiving Lithium therapy. The risk of Lithium discontinuation in case of side effects should be weighed against the psychological risk, and must be discussed with the psychiatrist.     

Long-term follow-up patients with leukemia receiving platelet transfusions: identification of a large group of patients who do not become alloimmunized

Alloimmunization is the major complication of platelet transfusion therapy in patients with acute leukemia. To evaluate whether alloimmunization continues to be a long-term problem in patients surviving induction therapy, 114 patients with acute nonlymphocytic leukemia (ANLL) who survived more than 6 mo and who received multiple courses of chemotherapy and abundant platelet transfusions were studied. Clinical response to random donor platelets and lymphocytotoxic antibody (LCTAb) were measured pretreatment and serially throughout the study period. Fourteen patients (12%) were alloimmunized upon admission, 34 (30%) patients became alloimmunized during remission induction therapy, and 66 (58%) patients did not become alloimmunized during that period. Sixty-one of these 66 patients (92%) never became alloimmunized and responded to random donor platelets during their subsequent course despite the fact they received multiple further platelet transfusions, whereas the alloimmunized patients tended to remain alloimmunized for their entire clinical course. There was no difference in age or sex between groups, and prognostic factors predicting alloimmunization could not be detected. In greater than 90% of patients not alloimmunized at admission, the presence or absence of LCTAb after induction predicts later alloantibody production. This information can be used to plan the type of platelet transfusions (HLA-matched or random donor) needed for subsequent maintenance and induction therapy. It may also help to identify a group of patients to whom more aggressive maintenance chemotherapy may be more safely administered.     

Mutations in the HIV type 1 integrase of patients receiving long-term dideoxynucleoside therapy do not confer resistance to zidovudine

Metabolites of AZT can inhibit HIV-1 integrase in vitro (Mazumder A, et al., Proc Natl Acad Sci USA 1994;91:5771-5775). To determine if long-term dideoxynucleoside therapy can lead to the emergence of HIV-1 AZT-resistant variants containing mutations in the integrase, we have sequenced the proviral DNA encoding the HIV-1 integrase of nine HIV-1-infected patients at different time points during treatment. Four of the nine patients developed mutations during the course of treatment. Although most mutations occurred at nonconserved amino acids, one patient developed a mutation at codon (R166T), a residue that is conserved among all integrases from known HIV-1 isolates. This mutation was introduced in the recombinant HIV-1 integrase protein to determine if it could confer resistance to AZT in vitro. We show that the R166T integrase mutant is still proficient at carrying 3'-processing and 3' end-joining but that the enzyme is not resistant to AZT-TP. Our results suggest that it is unlikely that integrase inhibition contributes to the antiviral activity of AZT.     

Mandatory reporting laws do not deter patients from seeking medical care.

STUDY OBJECTIVE: As of March 1994, 45 states had laws that, to varying extents, required health practitioners to report cases of domestic violence (DV). Colorado passed a mandatory DV reporting law in 1995. Laws that mandate police involvement in cases of DV injuries have been criticized because of concerns that these laws deter victims from seeking medical care. We hypothesized that these laws would deter DV victims from seeking medical care. METHODS: A questionnaire was administered in 3 stages: stage 1, convenience time blocks at 2 emergency departments and a primary care clinic; stage 2, prospective randomized blocks at an inner-city ED; and stage 3, a targeted population of women at risk for DV. All English-speaking, noncritical adult patients who presented during the time blocks were eligible to participate. RESULTS: Five hundred seventy-seven patients participated; 55% of the patients were aware of the mandatory DV reporting law. Twenty-seven percent of the patients would be more likely to seek medical care because of this law. Only 12% of patients stated that they would be less likely to seek medical care for a DV-related injury because of this law (15% of men and 9% of women; P =.001). There was no difference between ED patients and targeted female patients at risk for DV in seeking medical care ( P =.833). CONCLUSION: Only rarely did mandatory reporting laws appear to adversely affect a patient's decisions to seek medical care in this study. The benefits of mandatory reporting must be measured to assure that they justify deterrence to a small minority of patients.     

Acute lymphoblastic leukemia blast cells do not inhibit bone marrow hematopoietic progenitor colony formation

In newly diagnosed patients with acute lymphoblastic leukemia (ALL), bone marrow (BM) morphology always shows "replacement" by blasts with decreased or absent normal hematopoietic elements. To answer the question of whether ALL blasts inhibit replication and maturation of normal marrow progenitors, we studied the interaction of normal marrow with BM specimens from 16 new cases of ALL. Irradiated ALL blasts, supernatant derived from ALL blasts in suspension cultures, and conditioned medium prepared from ALL blasts augmented the colony-forming ability of normal marrow erythroid burst-forming unit (BFU-E), granulocyte-macrophage colony-forming unit (CFU-GM), megakaryocyte colony-forming unit (CFU-MK), and multilineage colony-forming unit (CFU-GEMM) progenitors. In sharp contrast to published data on the suppressive effects of acute myeloblastic leukemia cells on normal hematopoiesis in vitro, our results indicate that the growth advantage of ALL cells over normal marrow elements is not mediated through an inhibitory mechanism derived from leukemia cells.     

Isoflavonoids do not inhibit in vivo lipid peroxidation in subjects with high-normal blood pressure.

The isoflavonoids genistein and daidzein have been shown to have antioxidant activity in vitro, but their effects on in vivo oxidation have not been assessed. The newly described F2-isoprostanes are believed to currently represent the best available marker of in vivo lipid peroxidation. Therefore we have assessed the effects of a 55 mg daily isoflavonoid supplement on urinary F2-isoprostane concentrations in subjects with high-normal blood pressure (BP). A total of 59 subjects completed an 8-week parallel design, randomized, double blind, and placebo-controlled study. F2-isoprostanes, isoflavonoids and creatinine were measured in 24-h urine samples taken at baseline and at the end of the intervention. There were significant increases in urinary excretion of genistein (5.22+/-0.75 mg/day, P < 0.0001) and daidzein (2.53+/-0.43 mg/day, P < 0.0001) in the group taking the isoflavonoid supplement. Creatinine excretion was significantly correlated with F2-isoprostanes at baseline (r = 0.45, P < 0.01). After adjustment for baseline values, there was no significant difference between groups in creatinine adjusted post-intervention F2-isoprostane concentrations (P = 0.74). In addition, changes in genistein and daidzein excretion were not significantly correlated with changes in F2-isoprostanes in the isoflavonoid treatment group. These results are not consistent with the suggestion that the two soy derived isoflavonoids have in vivo antioxidant activity at a level of intake achievable by dietary means and in subjects with high-normal BP.     

Low-dose gold compounds inhibit fibroblast proliferation and do not affect interleukin-1 secretion by macrophages

We examined the effect of low concentrations of gold compounds on the proliferation of human fibroblasts. Gold sodium thiomalate (GST) inhibited both basal and interleukin-1-induced tritiated thymidine incorporation into fibroblasts in a dose- and time-dependent manner. Significant inhibition was observed at the level of 5 m̈g/ml GST, and >50% inhibition was attained at 10 m̈g/ml. These concentrations are attainable in the serum of treated patients. Similar inhibition was observed when <1 m̈g/ml auranofin, which is also within a serum-attainable range, was added. Low concentrations of GST (0–10 m̈g/ml) did not affect interleukin-1 secretion from lipopolysaccharide-stimulated human mononuclear phagocytes (Mϕ) when assessed by both human fibroblast and C3H/HeJ mouse thymocyte proliferation assays. When Mϕ precultured for 48 hours with GST (0–10 m̈g/ml) were added to the fibroblast culture in the presence or absence of lipopolysaccharide, there was no significant inhibition of Mϕ-induced DNA synthesis of fibroblasts. In contrast, when fibroblasts were precultured with GST (0–10 m̈g/ml) for 48 hours and freshly separated Mϕ were added, significant inhibition was observed in Mϕ-induced fibroblast proliferation at 5 m̈g/ml. These results suggest that low concentrations of GST directly cause a reduction of fibroblast proliferation, but do not affect the capability of Mϕ for induction of fibroblast proliferation. Therefore, gold compounds may play a role in the inhibition of the growth of rheumatoid pannus by direct inhibition of fibroblast proliferation.     

Passive immunization against insulin-like growth factor-I does not inhibit growth hormone-stimulated growth of dwarf rats.

Passive immunization against insulin-like growth factor-I (IGF-I) was undertaken in GH-deficient rats in an attempt to elucidate the relative importance of the endocrine vs.autocrine/paracrine actions of IGF-I in stimulating growth. Antiserum against IGF-I was raised in sheep and purified by affinity chromatography. The ability of the purified antibodies to neutralize the actions of IGF-I in vitro and bind IGF-I in vivo were extensively tested using L6 myoblast and cartilage bioassays. Four groups of male rats with isolated GH deficiency were used in the study. At 49 days of age the rats received 100 microliter normal saline given sc each day for 10 days, 2 mg/kg recombinant bovine GH (bGH) given in 100 microliter, sc, each day, 2 mg/kg bGH, sc, and 300 microliter immunoglobulin G purified from normal sheep serum given daily ip, or 2 mg/kg bGH plus 300 microliter anti-IGF-I immunoglobulin G daily, ip (a dose that was able to completely inhibit IGF-I actions on sulfate uptake into cartilage). Treatment with GH significantly increased growth rates (P less than 0.001) in the rats, but there was no difference between any of the three GH-treated groups; passive immunization against IGF-I did not diminish the GH-stimulated growth in these rats. Excess antibody could be detected in the plasma of all anti-IGF-I-treated rats at the conclusion of the experiment, and the antibody was capable of sequestering both free and binding protein-bound IGF-I. The absence of even a slight retardation of GH-stimulated growth in the anti-IGF-I-treated rats suggests that circulating IGF-I may not be important in mediating the growth-promoting actions of GH, although the immunoneutralization probably does not affect GH stimulation of tissue IGF-I production.