Transforming growth factor beta (TGF-beta), a potent inhibitor of erythropoiesis: neutralizing TGF-beta antibodies show erythropoietin as a potent stimulator of murine burst-forming unit erythroid colony formation in the absence of a burst-promoting activity

Transforming growth factor beta (TGF-beta) is a bifunctional regulator of the growth of myeloid progenitors and is here demonstrated to directly inhibit the growth of primitive erythroid progenitors by 95% to 100% regardless of the cytokines stimulating growth. Autocrine TGF- beta production of primitive hematopoietic progenitors has previously been reported. In the present study, a neutralizing TGF-beta antibody (anti-TGF-beta) added to serum-containing cultures, resulted in a 3-, 4- , and 25-fold increase in burst-forming unit erythroid (BFU-E) colony formation in response to interleukin-4 (IL-4) plus erythropoietin (Epo), SCF plus Epo, and IL-11 plus Epo, respectively. The growth of BFU-E progenitors has been suggested to require a burst-promoting activity in addition to Epo. Accordingly, we observed no BFU-E colony formation in serum-containing cultures in response to Epo alone. In contrast, 50 BFU-E colonies were formed when anti-TGF-beta was included in the culture. In serum-free cultures, Epo also stimulated BFU-E colony formation in the absence of other cytokines, whereas anti-TGF- beta had no effect on the number of colonies formed. Quantitation of TGF-beta 1 in serum by an enzyme-linked immunosorbent assay method showed predominantly the presence of precursor (latent) TGF-beta 1, but also showed active TGF-beta 1 at a concentration sufficient to potently inhibit erythroid colony formation. Thus, neutralization of active TGF- beta 1 in serum shows that Epo alone is sufficient to stimulate the growth of murine BFU-E progenitors.     

Fluorescent in vivo tracking of hematopoietic cells. Part I. Technical considerations

We report a new technology for in vivo tracking of hematopoietic cells, using fluorescent lipophilic probes. Because the probe is irreversibly bound in the lipids of the cell membrane; substantial numbers of dye molecules can be incorporated per cell and thus substantial signal to noise can be achieved. Although this technology can be used for all hematopoietic cells, these first findings are reported on red blood cells (RBCs) owing to the importance of the membrane to RBC function and integrity. We demonstrated that labeling 10% of the RBCs of a rabbit and reinjecting them into the animal makes possible the tracking of these cells at various times after injection. Furthermore, the labeling appears not to affect in vivo cell lifetime or cellular volume changes in response to hypotonic shock. The single cell fluorescence intensity of the labeled RBCs remains relatively constant for 60 days, and an immune response appears not to be generated against labeled cells. That labeled RBCs have lifetime kinetics in vivo, as shown in other studies, indicates that the membranes are functioning normally and are unaltered by the labeling technology. The technology we present is also applicable to white blood cells, bone marrow, and platelets.     

Determinants of impaired renal and vascular function are associated with elevated levels of procoagulant factors in the general population

Essentials

• Why venous thrombosis is more prevalent in chronic kidney disease is unclear.
• We investigated whether renal and vascular function are associated with hypercoagulability.
• Coagulation factors showed a procoagulant shift with impaired renal and vascular function.
• This suggests that renal and vascular function play a role in the etiology of thrombosis.

Summary

Background

Impaired renal and vascular function have been associated with venous thrombosis, but the mechanism is unclear.

Objectives

We investigated whether estimated glomerular filtration rate (eGFR), urinary albumin‐creatinine ratio (UACR), and pulse wave velocity (PWV) are associated with a procoagulant state.

Methods

In this cross‐sectional analysis of the NEO Study, eGFR, UACR, fibrinogen, and coagulation factors (F)VIII, FIX and FXI were determined in all participants (n = 6536), and PWV was assessed in a random subset (n = 2433). eGFR, UACR and PWV were analyzed continuously and per percentile: per six categories for eGFR (> 50^th [reference] to < 1st) and UACR (< 50^th [reference] to > 99th), and per four categories (< 50^th [reference] to > 95th percentile) for PWV. Linear regression was used and adjusted for age, sex, total body fat, smoking, education, ethnicity, total cholesterol, C‐reactive protein (CRP) and vitamin K antagonists use (FIX).

Results

Mean age was 55.6 years, mean eGFR 86.0 (12SD) mL 1.73 m^?² and median UACR 0.4 mg mmol^?1 (25th, 75th percentile; 0.3, 0.7). All coagulation factors showed a procoagulant shift with lower renal function and albuminuria. For example, FVIII was 22 IU dL^?1 (95% CI, 13–32) higher in the eGFR < 1st percentile compared with the > 50th percentile, and FVIII was 12 IU dL^?1 (95% CI, 3–22) higher in the UACR > 99th percentile compared with the < 50th percentile. PWV was positively associated with coagulation factors FIX and FXI in continuous analysis; per m/s difference in PWV, FIX was 2.0 IU dL^?1 (95% CI, 0.70–3.2) higher.

Conclusions

Impaired renal and vascular function was associated with higher levels of coagulation factors, underlining the role of renal function and vascular function in the development of venous thrombosis.

     

Discovery and characterization of a selective, nonpeptidyl thrombopoietin receptor agonist

OBJECTIVE: Peptide and other small molecule agonists have been described for several cytokines and growth factors. Hydrazone compounds described here as thrombopoietin receptor agonists were identified as activating STAT proteins in a Tpo responsive cell line. METHODS: STAT activation and analysis of signal transduction pathways in cell lines and normal human platelets was elucidated by Western blot and electrophoretic mobility shift assays. Proliferation assays in cell types responsive to other cytokines determined specificity for Tpo receptor. Flow cytometry quantified differentiation of CD34(+) cells into CD41(+) megakaryocytes and platelet production in vitro. RESULTS: Activation of STAT5, mitogen-activated protein kinase, p38, and early response genes by SB 394725 was similar to that induced by Tpo. SB 394725 induced a reporter gene response under a STAT activation promoter as well as the megakaryocyte-specific gpIIb promoter. The compound induced proliferation of Tpo responsive lines but demonstrated no activity in cell lines responding to other cytokines, i.e., erythropoietin, granulocyte-colony stimulating factor, interleukin-3, interferon-gamma. The response of normal human Tpo receptors was elucidated by measuring growth and differentiation of human bone marrow in vitro. Activation of endogenous Tpo receptors by SB 394725 was demonstrated in human and chimp platelets, but not in platelets of other species including mouse, dog, rabbit, or cynomolgus monkey. CONCLUSIONS: SB 394725, a small molecule with a molecular weight of 452 Da, is capable of activating Tpo-specific signal transduction, proliferation, and differentiation responses similar to the responses and functions of the protein growth factor, Tpo.     

Gonorrhoea in the asymptomatic patient: Presentation and the role of contact tracing for heterosexual men and women and for homosexual men

Summary Clinically silent gonorrhoea is the major problem in the control of the disease. Only 12 per cent of infected women reported in 1974 because of symptoms, compared with 97 per cent of infected heterosexual men and only 35 per cent of homosexual men with gonococcal proctitis alone. Homosexual men, compared with heterosexual men, had twice as many subsequent sexual contacts after infection and had a higher incidence of early syphilis. Eighty-four per cent had experienced passive anorectal intercourse. Ninety-seven per cent of men with gonococcal urethritis reported because of symptoms, but occasionally (particularly after unsuccessful treatment) urethral gonorrhoea in men may be clinically silent and even require tests of the overnight urethral secretion for diagnosis. For women, and for homosexual men who have had passive anorectal (or oral) intercourse, the indication for attendance for tests for gonorrhoea should be having run the risk, and not the presence of symptoms. Routine tests of the anorectum for gonorrhoea are essential in cases of women at risk, and for most homosexual men since over 80 per cent of these men will have had passive anorectal intercourse. Because gonococcal infections following treatment-failure are often clinically silent in both women and men, symptoms cannot be relied upon to indicate such failure. Follow-up smears and cultures are always essential.

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Kasuistik von Patienten mit asymptomatischer Gonorrhöe und die Bedeutung der durch Personen durchgeführten Kontaktermittlung bei heterosexuellen Männern und Frauen sowie bei homosexuellen Männern

Zusammenfassung Die asymptomatische Gonorrhöe ist das Hauptproblem bei der Kontrolle dieser Krankheit. Nur 12 Prozent der erfaßten infizierten Frauen im Jahre 1974 kamen wegen Beschwerden zur Untersuchung im Gegensatz zu 97 Prozent der infizierten heterosexuellen Männer und nur 35 Prozent der homosexuellen Männer mit isolierter gonorrhoischer Proktitis. Im Vergleich mit heterosexuellen Männern hatten homosexuelle Männer zweimal so viel sexuelle Kontakte nach der Infektion und eine höhere Inzidenz einer Syphilis im Frühstadium. 84 Prozent hatten passiven anorektalen Verkehr gehabt. 97 Prozent der Männer mit gonorrhoischer Urethritis meldeten sich wegen Beschwerden, aber gelegentlich (insbesondere nach unzureichender Behandlung) kann die gonorrhoische Urethritis bei Männern klinisch stumm sein und sie kann sogar Untersuchungen der nächtlichen urethralen Sekretion zur Diagnose erfordern. Für Frauen und für homosexuelle Männer, die passiven anorektalen (oder oralen) Verkehr gehabt haben, sollte der Grund ihres Kommens nicht das Vorhandensein von Symptomen sein, sondern ein mögliches eingegangenes Infektionsrisiko. Routine-Untersuchungen der Analgegend auf Gonorrhöe sind bei Frauen nach entsprechendem Geschlechtsverkehr unbedingt erforderlich, gleichfalls bei den meisten homosexuellen Männern, weil über 80 Prozent dieser Männer wohl einen passiven anorektalen Verkehr gehabt haben werden. Da Gonokokken-Infektionen nach Behandlungsversagen bei Männern und Frauen oft asymptomatisch sind, ist das Verschwinden der Symptome keine verläßliche Indikation für eine erfolgreiche Behandlung. Ständige Überwachung in Form von Abstrichen und kulturellen Untersuchungen ist auf alle Fälle erforderlich.

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This paper was presented in part on 27 September 1974 at a meeting of the British Society for Antimicrobial Chemotherapy.     

The Mr 95,000 gelatin-binding protein in differentiated human macrophages and granulocytes

Cultured adherent human macrophages and a promonocytic cell line, U 937, were previously shown to produce a Mr 95,000 gelatin-binding protein. The protein has no immunologic cross-reactivity with the well- characterized gelatin-binding protein fibronectin and the Mr 70,000 gelatin-binding protein produced by a variety of mesenchymal or epithelial cell types (T. Vartio et al, J Biol Chem 257:8862, 1982). In the present study the Mr 95,000 protein was found in Triton X-100 extracts of granulocytes purified from human blood buffy coat. The protein, as isolated by gelatin-agarose, was immunologically cross- reactive with the corresponding macrophage protein in immunoblotting assay. When peripheral blood and bone marrow cells were examined for the presence of the Mr 95,000 protein by indirect immunofluorescence, positive staining was detected only in differentiated granulocytes but not to any significant extent in metamyelocytes, myelocytes, promyelocytes, or in normal or leukemic blasts. In granulocytes the protein had a granular cytoplasmic distribution. In freshly prepared monocyte cultures, the Mr 95,000 protein was detected in low amounts in the cytoplasm, while along with differentiation of the cells into macrophages, the immunofluorescence increased in a reticular and vesicular cytoplasmic pattern and in a juxtanuclear cap, probably representing the Golgi complex. In conclusion, the Mr 95,000 gelatin- binding protein was specifically detected in macrophages and granulocytes and may thus serve as a differentiation marker for these phagocytic cells.     

Identification of altered dipeptidyl-peptidase activities as potential biomarkers for unipolar depression

Background

Changes in circulatory aminopeptidases [dipeptidyl-peptidase-IV (DPP-IV), Prolyl-oligopeptidase (POP) and Leucine aminopeptidase (LAP)] activities have been found to be associated with psychiatric illnesses and inflammatory diseases.

Methods

The discriminatory indices of aminopeptidases activities were assessed by enzymatic assays in plasma samples from 240 unipolar depression (UD) patients and 264 matched controls. In addition the relationship between soluble and cellular DPP-IV activity was determined in plasma and blood cells from healthy subjects.

Results

Greater than 95% of the plasma DPP-IV activity could be blocked by inhibitors, demonstrating the specificity of the assay. Also, DPP-IV protein and activity levels were strongly correlated. In contrast, only 50% of the membrane-bound activity in blood cells was inhibited, which suggested that other similar peptidases may be present in these cells. UD patients had decreased plasma levels of DPP-IV and POP activities compared to healthy controls with a concomitant increase in LAP activity. Finally, testing of the LAP/DPP-IV ratio resulted in good discrimination of UD patients from controls with an area under the curve—receiver operating characteristic of 0.70.

Limitations

Further biological validation studies using different cohorts are warranted.

Conclusions

The finding that plasma DPP-IV activity was decreased and LAP activity was increased in UD patients suggests the potential value for testing the levels of these enzymes for improved classification of patients. In addition, the changes in these enzymes, suggests that the proteolytic maturation of their proneuropeptide and prohormone subtrates may also be affected in UD, resulting in altered production of the associated bioactive peptides.