HLA antigens on platelet membranes. In vitro and in vivo studies

In order to determine whether HLA-A,B antigens of platelets are integral membrane constituents or rather represent adsorbed plasma proteins, their presence in plasma and their adsorbability onto platelet membranes was studied by in vitro and in vivo experiments. The amount of HLA antigens was quantitated by inhibition of lymphocytotoxicity (LCT) and by enzyme-linked immunosorbent assay (ELISA) using operationally monospecific polyclonal HLA antibodies or murine HLA-specific monoclonal antibodies, respectively. We found that in 11 out of 13 HLA-A2 and in 9 out of 10 HLA-B13 experiments, platelets from antigen-negative donors pretreated with plasma from the same number of antigen-positive donors inhibited LCT to the same extent as platelets from antigen-positive donors. Nevertheless, the in vitro adsorbed HLA antigens onto antigen-negative platelets were, unlike those on antigen-positive platelets or in plasma, not reactive with monoclonal antibodies as quantitated by ELISA. Similarly, infusion of HLA-A2-negative platelets from single donors into 3 HLA-A2-positive, thrombocytopenic patients with bone marrow failure led to a good platelet increment, but did not convert the HLA type of donor platelets, neither at 2 h nor at 18 h posttransfusion. On the basis of these results, we conclude that soluble HLA antigens can be taken up by human platelets from plasma in small amounts. However, the major portion of HLA antigens appears to be integral membrane constituents.     

Serotonin induced platelet aggregation in systemic sclerosis

We studied in vitro platelet aggregation response to serotonin in 19 patients with systemic sclerosis and 18 matched controls. Individuals with early systemic sclerosis had increased % platelet aggregation at 3 minutes over a wide range of serotonin concentrations when compared to individuals with late systemic sclerosis. Patients with generalized disease did not differ from those with limited disease. No significant differences in initial slope of platelet aggregation were detected in any patient subgroup. These data support previous studies suggesting platelet activation in systemic sclerosis and further suggest that future studies of platelet abnormalities in systemic sclerosis should consider the effects of disease duration.     

In vivo and in vitro evidence of B cell hyperactivity during Lyme disease

In vitro IgM production by cells from patients with Lyme disease rose during the illness in those studied soon after onset, but fell from elevated levels in those initially studied later than 1 week after onset. Borrelia burgdorferi stimulated normal and patients' cells produced IgM, with cells from acutely ill patients producing the most; production fell during convalescence. Patients with active Lyme disease and those destined for later manifestations often had serum agglutinins for heterologous red blood cells. Thus, there is in vitro and in vivo B cell hyperactivity in Lyme disease caused by B. burgdorferi. Both mitogenic and antigenic stimulation of B cells may induce the humoral response seen in complicated Lyme disease.     

In vitro inhibition of hematopoiesis in a patient with systemic sclerosis treated with D-penicillamine.

A 66-year-old Japanese woman with severe scleroderma developed anemia and thrombocytopenia due to D-penicillamine (D-Pen) treatment, although the leukopenia was not markedly severe. Cessation of D-Pen and the start of corticosteroid therapy were followed by recovery from bicytopenia. We examined the in vitro inhibition of the clonogenic capacity of bone marrow hematopoietic progenitor cells of this patient by D-Pen, and found strong inhibition in burst-forming unit-erythroid and colony-forming unit-megakaryocyte assays, but not in colony-forming unit-granulocyte/macrophage assays. These findings suggest that bicytopenia in this patient was caused by D-Pen and may be due to different sensitivities in the hematopoietic lineage.     

Diastolic abnormalities in systemic sclerosis: evidence for associated defective cardiac functional reserve.

OBJECTIVE: To investigate the pattern of diastolic abnormalities in patients with systemic sclerosis (SSc) and the relationship between impaired ventricular filling and systolic function. METHODS: Twenty four patients with SSc underwent M-mode and two dimensional echocardiography using echo-Doppler and gated blood pool cardiac angiography, both at rest and after exercise. RESULTS: An impaired diastolic relaxation of the left ventricle was detected in 10 of the 24 patients with SSc. Left ventricular ejection fraction at rest in these 10 patients with impaired ventricular filling did not differ from that in the remaining 14 patients, but eight of the 10 failed to increase their ejection fraction during exercise, compared with two of the 14 with normal ventricular filling (p = 0.003). CONCLUSION: Impaired relaxation of the left ventricle is a recently described feature of scleroderma heart disease. Diastolic dysfunction in SSc could depend on myocardial fibrosis or myocardial ischaemia, or both. It was found to be associated with a defective cardiac functional reserve. However, its prognostic significance remains to be clarified.     

Comparative studies of esophageal function in systemic sclerosis.

Three modalities for assessing esophageal dysfunction in patients with systemic sclerosis were prospectively compared. Seventeen patients underwent (a) esophageal manometry with measurement of distal esophageal peak contraction pressure amplitude, percentage of peristaltic waves, and lower esophageal sphincter pressure; (b) cine-esophagography with scoring based on residual contrast and the character of visualized waves; and (c) esophageal transit scintigraphy with quantification of residual swallowed tracer. Highly significant correlations were found between scintigraphic residual and cine-esophagography score, between scintigraphic residual and manometric amplitude, and indeed between all pairs of measured esophageal function parameters except those involving lower esophageal sphincter pressure. In addition, scintigraphy and cine-esophagography showed comparable ability to discriminate between patients with abnormal and normal esophageal motor function. Symptoms did not significantly correlate with quantitative parameters, nor did they have diagnostic discriminating ability. Induction of Raynaud's phenomenon in a subgroup of patients had no detectable effect on esophageal function. It was concluded that these three diagnostic modalities are approximately equivalent in their ability to detect esophageal dysmotility in systemic sclerosis and measure its severity.     

Vascular damage and lack of angiogenesis in systemic sclerosis skin

The aim of this study was to analyse microvascular damage and compensatory angiogenesis in skin from patients with systemic sclerosis (SSc) compared with systemic lupus erythematosus (SLE), Raynaud's phenomenon (RP) and healthy controls. Immunohistochemistry was used for skin biopsies (9 SSc, 10 SLE, 9 RP and 12 healthy controls) using von Willebrand factor and ₃ integrin subunit specific antibodies, TechMate immunostaining robot and biotin-streptavidin protocol. In the early stages of SSc, vWF was found in the perivascular space and interstitial matrix in papillary but not in the reticular dermis, in particular around small oedematous blood vessels infiltrated by mononuclear cells. The extravascular release of vWF in SSc specimens was associated with weak or even a total lack of immunoreactivity within the associated endothelial cells. Late stages of SSc were characterised by loss of the dermal papillae, subepidermal fibrosis, hypovascularity and strong endothelial vWF expression without extravascular leakage. In all SSc patients studied only a few vascular profiles were weakly immunostained for ₃ integrin subunit. This work demonstrates that vWF is not only released into the systemic circulation, but is also leaked to the perivascular space/matrix. This local release and deposition of vWF is probably a sensitive and early marker of microvascular involvement in SSc pathogenesis. Local vWF release may play a role in platelet adhesion, aggregation, thrombogenesis and dermal connective tissue remodelling. In spite of some attempts towards compensatory angiogenesis in SSc, as evidenced by ₃ integrin subunit expression, it was evident that the angiogenic response was not able to prevent the development of hypovascularity during the advanced stages of the disease.     

In vitro platelet adhesion to vascular subendothelium: studies in patients with polycythaemia vera

In this work we studied platelet adhesion to subendothelial surfaces in 10 patients with polycythaemia vera and 10 healthy volunteers at 40% Hct (corresponding to the mean value of our control group) and 55% Hct (a value roughly corresponding to the mean Hct in polycythaemic patients). Platelet concentration was kept constant at 2.0-2.5 X 10(11)/l. The results indicate that there was a statistically significant increase in adhesion both in controls and in patients with Hct varying from 40% to 55%. The contribution of the higher Hct in promoting platelet adhesion was comparable in the two groups. When red blood cells (RBC) from the patients were tested with platelets from healthy volunteers in cross-over experiments, they promoted adhesion in the same way as control RBC. Similarly, when patients' platelets were mixed with control RBC, adhesion was the same as control platelets. These data indicate that platelet and RBC contribution to this parameter are not significantly modified in this group of polycythaemic patients, provided that platelet and RBC values are adjusted to control range.     

In vitro studies of the effect of buflomedil on platelet responsiveness

The effect of buflomedil (Fonzylane; Laboratoire Lafon, Maisons-Alfort, France) on platelet function, a drug used clinically for the treatment of peripheral vascular diseases, was investigated in vitro. The compound significantly inhibits epinephrine-induced aggregation at the micromolar level. At higher doses (approximately 1 mM), a weak inhibition of ADP- and collagen-induced aggregation was observed; at these concentrations, buflomedil inhibits granular secretion and the interaction of fibrinogen with its receptor on platelet. Further investigations indicate that the drug affects calcium uptake at the membrane level and inhibits the binding of [3H]-yohimbine to the same extent as observed with phentolamine. The IC50 determined from competition binding assays was 1 +/- 0.5 microM. This value was consistent with the affinity constant approximated for the binding of [3H]-buflomedil to non-stimulated platelets. Taken-together, these results indicate that the vasoactive compound buflomedil is a weak antiaggregating agent which exhibits alpha 2-adrenergic antagonistic properties.     

In vitro studies of lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is associated with abnormal airway smooth muscle that leads to the characteristic pathology of lung nodule formation and destruction of lung tissue. The current authors have previously identified abnormal behaviour of airway smooth muscle cells from patients with asthma. In this study, cells and tissue sections derived from patients with LAM (n=7), asthma (n=8), and nonasthmatic controls (n=9) were compared. The presence of the antigen human melanosome (HM)B-45 was investigated, along with the proliferation and release of extracellular matrix proteins, release of endogenous prostaglandin E2 (PGE2), vascular endothelial growth factor and connective tissue growth factor, and the expression of integrins. Positive HMB-45 staining was found in all LAM patients and no controls. Proliferation of LAM cells was not different from control cells nor was its inhibition by beta-agonists, corticosteroids, rapamycin or PGE2. However, endogenous PGE2 levels were markedly decreased in LAM cells, and this was associated with decreased expression of the inducible form of cyclooxygenase (COX-2). The increased levels of connective tissue growth factor seen in asthma cells were not observed in LAM. Elastin mRNA in response to transforming growth factor-beta stimulation was markedly lower in LAM cells than either asthma or control cells. In conclusion, lymphangioleiomyomatosis cells exhibit abnormal properties in vitro that may contribute to pathophysiology and symptomatology in patients with lymphangioleiomyomatosis.     

Ferroportin: lack of evidence for multimers

Ferroportin is a multi-transmembrane glycoprotein that mediates iron export from cells. Mutations in ferroportin are linked to type IV hemochromatosis, a dominantly inherited disorder of iron metabolism. Multimers of ferroportin, whose existence may relate to the dominant inheritance pattern of disease, have been detected in some studies but not others. We looked for evidence of multimerization in several different types of experiment. We assayed the maturation of mutant and wild-type ferroportin and found that loss-of-function mutants had a reduced half-life but did not alter the stability of coexpressed wild-type. Using bioluminescence resonance energy transfer analysis, we tested how mature wild-type ferroportin behaved in intact live cell membranes. Ferroportin–ferroportin interactions gave the very low acceptor/donor ratio-independent energy transfer levels characteristic of random protein–protein interactions, consistent with ferroportin behaving as a monomer. Consistent with these experiments, we were unable to detect a dominant negative functional effect of mutant ferroportin on wild-type, even when expression of wild-type protein was titrated to low levels. These data suggest that dominantly inherited ferroportin disease does not result from the direct action of a mutated protein inhibiting a wild-type protein within multimers. We propose other possible mechanisms of disease.     

111In platelet imaging of left ventricular thrombi. Predictive value for systemic emboli

To determine whether a positive indium 111 platelet image for a left ventricular thrombus, which indicates ongoing thrombogenic activity, predicts an increased risk of systemic embolization, we compared the embolic rate in 34 patients with positive 111In platelet images with that in 69 patients with negative images during a mean follow-up of 38 +/- 31 (+/- SD) months after platelet imaging. The positive and negative image groups were similar with respect to age (59 +/- 11 vs. 62 +/- 10 years), prevalence of previous infarction (94% vs. 78%, p less than 0.05), time from last infarction (28 +/- 51 vs. 33 +/- 47 months), ejection fraction (29 +/- 14 vs. 33 +/- 14), long-term or paroxysmal atrial fibrillation (15% vs. 26%), warfarin therapy during follow-up (26% vs. 20%), platelet-inhibitory therapy during follow-up (50% vs. 33%), injected 111In dose (330 +/- 92 vs. 344 +/- 118 microCi), and latest imaging time (greater than or equal to 48 hours in all patients). During follow-up, embolic events occurred in 21% (seven of 34) of patients with positive platelet images for left ventricular thrombi as compared with 3% (two of 69) of patients with negative images (p = 0.002). By actuarial methods, at 42 months after platelet imaging, only 86% of patients with positive images were embolus free as compared with 98% of patients with negative images (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of platelet aggregability by losartan in essential hypertension

Most clinical events associated with hypertension have a thrombotic component. Losartan is a selective, competitive antagonist of the thromboxane A2 receptor in experiments performed in isolated vascular strips and in human and rat platelet-enriched plasma. In this study, we investigated for the first time whether losartan at therapeutic doses has an effect on platelet aggregability and indexes of fibrinolysis in essential hypertensive subjects. Changes in the dose-response curve to platelet aggregation induced by the thrombin receptor-activating peptide SFLRRN-NH2 were determined in 9 patients (56% men, 72% white; mean age 52.8 years) with stage I or II essential hypertension and in 9 untreated healthy volunteers. After a 4-week washout period, hypertensive subjects received 2 weeks of placebo followed by 4 weeks of losartan 50 mg/day. Both subjects and end points were blinded for treatment assignment. In addition, plasminogen activator inhibitor type 1 antigen and von Willebrand antigen were studied in all patients and controls. Four weeks of losartan produced a statistically significant (p <0.05) increase in the concentration of SFLRRN-NH2 required to induce a half-maximal response in platelet aggregation extent and rate 4 weeks after initiation of treatment. The decrease in platelet aggregability was independent of blood pressure control and the effects of gender and age. Losartan had no effect on plasma concentrations of plasminogen activator inhibitor-1 and von Willebrand factor in hypertensive subjects. These data demonstrate for the first time a novel antiplatelet effect of losartan at therapeutic doses, which was independent of changes in blood pressure, plasma markers of fibrinolytic activity, and endothelial perturbation.     

The microbiome and systemic sclerosis: A review of current evidence

Systemic sclerosis (SSc) is characterized by immune dysregulation, vasculopathy, and fibrosis of multiple organs. The gastrointestinal (GI) tract is the most common internal organ manifestation, which contributes to significant morbidity and mortality in patients with SSc. Emerging reports have identified unique microbial taxa alterations in the GI microbiome of patients with SSc as compared to healthy controls (HC). These taxa alterations include differences at the phyla (e.g., Bacteroidetes) and genera (e.g., Bacteroides, Clostridium, Faecalibacterium, and Lactobacillus) level. In addition, some genera have been associated with more severe GI symptoms (e.g., Prevotella and Akkermansia). This review summarizes the current evidence on factors influencing the GI microbiome, GI microbiome alterations in SSc as compared to HC, and in SSc subgroups according to disease manifestations. Current exploration in therapeutic interventions that target the GI microbiome is discussed.     

Bacterial vaginosis in lesbians: evidence for lack of sexual transmission.

The effect of non-heterosexual factors on the vaginal flora has been studied. Ninety-one lesbians attending a specialist genitourinary medicine service for lesbians were studied. Bacterial vaginosis (BV) was diagnosed in 51.6% of them. While most of the women had previously had a male sexual partner, the presence of BV was not associated with a male sexual partner in the previous 12 months. A detailed analysis of lesbian sexual practices in the group did not relate BV to any sexual practice which would have the propensity to pass vaginal secretions from one to the other.     

Radionuclide esophageal transit studies in progressive systemic sclerosis: an analysis of longitudinal data.

Nineteen patients with progressive systemic sclerosis (PSS) were studied by radionuclide esophageal transit (ET) and followed longitudinally for 3 to 5 years. Results were expressed as percent retention at 20 s and 10 min. There was gradual deterioration of ET at both 20 s and 10 min. When results were grouped into quartiles, deterioration occurred in 58.5% of followup studies in patients who initially had potential to deteriorate regarding 20 s retention and in 48% of similar patients regarding 10 min retention.     

Platelet alpha 2 adrenoceptor characteristics during the morning increase in platelet aggregability.

The incidence of myocardial infarction and sudden cardiac death increases in the morning, as do platelet aggregability and sympathetic activity. We considered the possibility that increased platelet aggregability in the morning was due to an increase in platelet alpha 2 adrenergic receptor number or agonist binding affinity, or to a temporal disparity between the increase in sympathetic activity that accompanies arising and the anticipated decrease in platelet high affinity alpha 2 adrenoceptors. To evaluate these possibilities, we studied eight healthy male volunteers (20-35 years) before and 1-1.5 and 3-4 h after they arose at 08.00 h and performed routine morning activities. Platelet aggregability was determined in platelet-rich plasma; alpha 2 adrenoceptor density and agonist binding affinity were determined in intact platelets and in membrane preparations using the alpha 2 selective ligand [3H]-yohimbine. The threshold concentration of epinephrine required to produce biphasic aggregation decreased (i.e. platelet aggregability increased) from 3.6 +/- 1.2 to 0.9 +/- 0.3 microM (P less than 0.05) after arising. However, alpha 2 adrenoceptor density (380 +/- 71 to 365 +/- 51 sites per platelet) and agonist binding affinity assessed simultaneously did not change after arising, suggesting that the increase in platelet aggregability is due to factors extrinsic to the platelets or to an intra-platelet mechanism distal to the receptor level.