Transient deficiency of peripheral blood accessory cells in supporting T cell mitogenesis in patients suffering from chronic idiopathic thrombocytopenic purpura after intravenous gammaglobulin treatment

Mitogenic response of blood lymphocytes to phytohemagglutinin (PHA) and to OKT3 monoclonal antibodies was investigated in 7 patients suffering from chronic idiopathic thrombocytopenic purpura (ITP) before, during and after high-dose intravenous (i.v.) immunogammaglobulin (IgG) infusion. The platelet count rose above the pre-treatment values during infusion therapy in all patients but one. Five out of seven patients presented elevated platelet-associated IgG (PA-IgG) levels at the time of the first infusion; four of these showed an increase in platelet count and a transient reduction or normalization of PA-IgG after IgG infusion. Five out of seven patients showed an impairment of T lymphocyte mitogenic response to PHA and OKT3 before therapy. All patients responded to IgG therapy with a transient deficiency of FcR mediated monocytes (Mo) in supporting T cell mitogenesis induced by both mitogens during and after IgG infusion. This reduced cooperative capability of Mo disappeared at various times after the end of therapy (range 3-12 days). The transient alteration of Mo function, possibly due to a modification in the surface number or in the affinity of Fc-receptors, can explain in part, the increase in platelet count during and after IgSRK infusion.     

Efficacy of rhesus antibodies (Anti-Rh0 (D)) in autoimmune thrombocytopenia: correlation with response to high dose IgG and the degree of haemolysis

We have compared the efficacy of high-dose IgG with that of Rhesus antibodies (anti-Rh0 (D)) in 5 patients with autoimmune thrombocytopenic purpura (3 adults and 2 children). Although only transient, high-dose IgG (0.4 g/kg X 5 days) was effective in all patients (peak values 50-200 X 10(9)/1), whereas anti-Rh0 (D) (11-20 micrograms/kg X 5 days) led to comparable results in only 3 patients (165 X 10(9)/1, 72 X 10(9)/1, 33 X 10(9)/1). This response to anti-Rh0 (D) was neither related to the degree of induced haemolysis (increase of LDH and decrease of haptoglobin) nor to the amount of IgG antibodies bound to red blood cells, as quantified by the 125-I-antiglobulin test. A decrease of platelet-associated IgG was recorded in 3 patients: 2 of them showed an improvement of platelet counts and in one of them there was no response. We conclude that the therapeutic response of high-dose IgG and anti-Rh0 (D) is independent of the degree of induced haemolysis and may not be predicted from the effectiveness of either therapy alone.     

Prediction of the effect of immunoglobulin therapy in ITP

The correlation between the response to high-dose immunoglobulin therapy (IVIg) and the sequestration pattern of Indium-labeled platelets (In-PLT) in the body was studied in 9 patients with chronic idiopathic thrombocytopenic purpura (ITP). Patients that has prominent platelet sequestration in the spleen responded to IVIg. In these patients, splenic sequestration decreased by 20-30% after IVIg without significant changes in hepatic sequestration. This finding suggests that the blocking of splenic Fc receptors with immunoglobulin minimized the destruction of sensitized platelets. However, patients who had almost equal platelet sequestration in the liver and spleen did not respond to IVIg. In these patients, hepatic sequestration decreased after IVIg, whereas splenic sequestration increased. Thus, it appears that estimating the platelet sequestration pattern using In-PLT is useful for predicting the effects of IVIg.     

Nematode-Induced Interference with Vaccination Efficacy Targets Follicular T Helper Cell Induction and Is Preserved after Termination of Infection

One-third of the human population is infected with parasitic worms. To avoid being eliminated, these parasites actively dampen the immune response of their hosts. This immune modulation also suppresses immune responses to third-party antigens such as vaccines. Here, we used Litomosoides sigmodontis-infected BALB/c mice to analyse nematode-induced interference with vaccination. Chronic nematode infection led to complete suppression of the humoral response to thymus-dependent vaccination. Thereby the numbers of antigen-specific B cells as well as the serum immunoglobulin (Ig) G titres were reduced. T_H2-associated IgG1 and T_H1-associated IgG2 responses were both suppressed. Thus, nematode infection did not bias responses towards a T_H2 response, but interfered with Ig responses in general. We provide evidence that this suppression indirectly targeted B cells via accessory T cells as number and frequency of vaccine-induced follicular B helper T cells were reduced. Moreover, vaccination using model antigens that stimulate Ig response independently of T helper cells was functional in nematode-infected mice. Using depletion experiments, we show that CD4⁺Foxp3⁺ regulatory T cells did not mediate the suppression of Ig response during chronic nematode infection. Suppression was induced by fourth stage larvae, immature adults and mature adults, and increased with the duration of the infection. By contrast, isolated microfilariae increased IgG2a responses to vaccination. This pro-inflammatory effect of microfilariae was overruled by the simultaneous presence of adults. Strikingly, a reduced humoral response was still observed if vaccination was performed more than 16 weeks after termination of L. sigmodontis infection. In summary, our results suggest that vaccination may not only fail in helminth-infected individuals, but also in individuals with a history of previous helminth infections.     

Release of platelets into the circulation induced by gamma globulin treatment in a case of idiopathic thrombocytopenic purpura

The kinetics and distribution in vivo of 111In-labelled platelets prior to and during intravenous high-dose gamma globulin therapy in a 3-months-old child with idiopathic thrombocytopenic purpura were evaluated. Concomitantly with a prompt increase in platelet concentration we found evidence for release of platelets into the circulation, presumably to a large extent originating from the spleen and liver. Our results support the concept that, in response to high-dose gamma globulin therapy, platelets are not only forced to remain in but are also released into the circulation, presumably as a result of mobilization of platelets adhering to cells of the monocyte-macrophage system.     

The comparison of gammaglobulin to steroids in treating adult immune thrombocytopenia. An interim analysis

Symptomatic immune thrombocytopenia is a life-threatening situation which is conventionally treated in the adult with prednisone, although subsequent splenectomy is frequently unavoidable. Recently, high-dose intravenous gamma-globulin has been reported to be an effective alternative option, particularly in children. To determine the role of this agent in adults a controlled prospective trial has been undertaken. Previously untreated patients with immune thrombocytopenia were randomised to compare oral prednisone (1 mg/kg/day: Group 1: n = 13) to high-dose intravenous gamma-globulin (400 mg/kg on days 1 through 5: Group 2: n = 7), or a combination of both agents given on the same schedule (Group 3: n = 12). The time from diagnosis to commencement of treatment, initial platelet counts, age and sex were comparable in the three groups. At this interim analysis there has been no mortality, but one patient has suffered a cerebrovascular accident. Objective response, defined as a platelet count greater than 50 x 10(9)/l, was achieved in a median of 5, 5 and 3 days, whereas the time taken to reach peak counts were 9, 5 and 7 days, respectively. The relapse rates, percentage of patients subsequently requiring splenectomy for control of symptomatic bleeding and the postoperative course was comparable between the three groups. These data, although preliminary, re-emphasize differences between the paediatric and adult forms of immune thrombocytopenia and also suggest in the latter patients a need for caution before advocating replacement of prednisone by gammaglobulin as the primary form of treatment.     

In vitro functions of lymphocytes during high-dose medroxyprogesterone acetate (MPA) treatment

Summary The number and function of T and B lymphocytes were studied in endometrial cancer patients during 3 months' treatment with high-dose medroxyprogesterone acetate (MPA). No significant changes were observed in the proportions of T and B cells or in the function of B cells during MPA treatment. At 3 months, lymphocyte blastogenic responsiveness to mitogens was slightly reduced both in patients treated with standard therapy alone (intracavitary radium and surgery) and in patients receiving additional MPA therapy. These results indicate that other factors than progesterone are responsible for the suppression of lymphocyte blastogenesis induced by mitogens during high-dose progestin treatment.     

Parasitism, immune response and reproductive success in the house martin Delichonurbica

Parasites often exert strong selection pressures on their hosts that have evolved anti-parasite defences to counter the negative effects of parasites. We studied the relationship between intensity of parasitism, one aspect of host immune response, and host reproductive success, using the house martin bug Oeciacushirundinis and its house martin Delichonurbica host as a model system. Experimental manipulation of parasite load of nests during laying of the first clutch altered the intensity of parasitism. Parasites reduced the reproductive success of their hosts measured in terms of body condition and survival of nestlings. Host immune response, measured as the concentration of gammaglobulins and total plasma proteins, was positively associated with parasite reproduction, estimated as the number of juvenile parasites, but was only weakly related to the intensity of adult parasites. The concentration of gammaglobulins was negatively related to nestling body mass, implying a trade-off between immune function and body condition. Parasite reproduction thus exerts a cost on hosts by increasing the immune response. Received: 25 August 1997 / Accepted: 3 November 1997     

Gray platelet syndrome: selective alpha-granule deficiency and thrombocytopenia due to increased platelet turnover

Clinical and laboratory studies of two siblings, both suffering from gray platelet syndrome (GPS) are described. The patients had a mild bleeding disorder, their platelets were blue-gray in panoptic stains, and alpha-granules were markedly reduced, as shown by electron microscopy. The platelet content of platelet factor 4 and that of beta-thromboglobulin were significantly reduced (3%-7% of normal). Platelet count was decreased (33-150 X 10(9)/1) and small platelets were increased in platelet volume distribution. Bleeding time was prolonged on most occasions. Bone marrow aspiration was performed in one patient and revealed increased reticulin fibers, however, megakaryocyte count was normal. The mean platelet survival was 4.8 days using 111indium-labelled platelets. In this patient, platelet-associated IgG was within the normal range. Prednisone therapy failed to increase platelet count. Dental surgery was performed under cover of desmopressin and no bleeding complication occurred; however, no improvement of bleeding time was observed. The patient delivered a healthy male infant without hemorrhaging while under concurrent platelet transfusion therapy.     

High-dose intravenous therapy with immune globulin before delivery for idiopathic thrombocytopenic purpura.

A 15-year-old girl with a 9-year history of idiopathic thrombocytopenic purpura resistant to high-dose steroid therapy and to splenectomy was admitted to hospital at 35 weeks'' gestation with a platelet count of 10 X 10(9)/L. The bleeding time was normal, and measures of platelet aggregation were nearly so. Treatment with high intravenous doses of polyvalent immune globulin led to a rise in the platelet count to more than 110 X 10(9)/L within 5 days. An elective cesarean section was performed through the lower uterine segment with good hemostasis. After delivery the platelet count fell to its former level, but no postpartum bleeding occurred. There was a brief episode of thrombocytopenia in the infant, with some petechiae but no other hemorrhagic manifestations. No untoward effects of the immune globulin infusion were observed in either mother or daughter.     

Immune response to CagA protein is associated with improved platelet count after Helicobacter pylori eradication in patients with idiopathic thrombocytopenic purpura

BACKGROUND: Improvement in platelet counts has been reported after eradication of Helicobacter pylori in patients with idiopathic thrombocytopenic purpura (ITP). We examined the levels of serum markers of gastritis and anti-CagA (cytotoxin-associated gene A) IgG antibody in patients with ITP to investigate whether these factors are associated with the platelet response after H. pylori eradication therapy. MATERIALS AND METHODS: One hundred and sixteen consecutive patients with ITP were assessed for H. pylori infection by (13)C-urea breath test and serum H. pylori antibody test. Patients with H. pylori infection received eradication therapy. Before and after eradication therapy, we evaluated serum levels of gastrin, pepsinogen (PG)-I, and PG-II and the anti-CagA IgG antibody titer. RESULTS: H. pylori infection was found in 67 (58%) of the 116 patients with ITP. Fifty-two infected patients received eradication therapy, which was successful in 44 patients (85%). Twenty-seven patients (62%) showed an increased platelet count and were identified as responders. The duration of ITP was shorter in responders than in nonresponders (p = .017). There was no difference of the levels of gastrin and PGs between responders and nonresponders. Before eradication therapy, the serum anti-CagA antibody titer did not differ significantly between responders and nonresponders. However, reduction in the anti-CagA antibody titer after eradication therapy was significantly greater in responders than in nonresponders (p = .013). CONCLUSIONS: H. pylori eradication therapy improves the platelet count in H. pylori-positive patients with ITP of short duration. Immune response of hosts to CagA protein of H. pylori may play a role in the pathogenesis of ITP.     

Platelet sparing effect of COX II inhibition used with pegylated interferon alfa-2a for the treatment of chronic hepatitis C: a short term pilot study

The role of a cyclooxygenase (COX) II inhibitor in reducing microvascular inflammation and the platelet count associated with interferon (IFN) plus ribavirin therapy of chronic hepatitis C (HCV) was assessed. Three plasma mediators (biomarkers) associated with platelet activation, inflammation and fibrosis were measured. Eighteen IFN na?ve patients were studied. Nine were treated with pegylated IFN alfa-2a (PEG-IFN alpha-2a) plus ribavirin and rofecoxib; nine were treated with PEG-IFN alpha-2a plus ribavirin. A complete blood count, liver panel and HCV-RNA were assayed weekly. Human soluble P-selectin (hs-P-selectin), human interleukin-8 (IL-8), human interleukin-13 (IL-13) and human thrombopoietin (TPO) were assayed at 4 week intervals. The COX II inhibitor reduced the platelet reduction experienced with PEG-IFN alpha-2a treatment of HCV despite a reduction in the plasma TPO level. Hs-P-selectin was increased in both groups. In contrast, human IL-8 levels declined to undetectable levels in virologic responders. Similarly, human IL-13 levels declined with therapy (P < 0.001). These data suggest that: (1) a COX II inhibition is associated with an increase in the platelet count despite a reduction in the TPO level; (2) human IL-8 and human IL-13 but not hs-P-selectin levels decline in those who experience an early virologic response.     

Comparison of Aggregometry with Flow Cytometry for the Assessment of Agonists′-Induced Platelet Reactivity in Patients on Dual Antiplatelet Therapy

Data on the agreement between aggregometry and platelet activation by flow cytometry regarding the measurement of on-treatment platelet reactivity to arachidonic acid (AA) and adenosine diphosphate (ADP) are scarce. We therefore sought to compare three platelet aggregation tests with flow cytometry for the assessment of the response to antiplatelet therapy. Platelet aggregation in response to AA and ADP was determined by light transmission aggregometry (LTA), the VerifyNow assays, and multiple electrode aggregometry (MEA) in 316 patients receiving aspirin and clopidogrel therapy after angioplasty with stent implantation. AA- and ADP-induced P-selectin expression and activated glycoprotein (GP) IIb/IIIa were determined by flow cytometry. LTA, the VerifyNow P2Y₁₂ assay and MEA in response to ADP correlated significantly (all p<0.001), and the best correlation was observed between LTA and the VerifyNow P2Y₁₂ assay (r = 0.63). ADP-induced platelet reactivity by all aggregation tests correlated significantly with ADP-induced P-selectin expression and activated GPIIb/IIIa (all p<0.001). The best correlation was seen between the VerifyNow P2Y₁₂ assay and activated GPIIb/IIIa (r = 0.68). The platelet surface expressions of P-selectin and activated GPIIb/IIIa in response to ADP were significantly higher in patients with high on-treatment residual platelet reactivity (HRPR) to ADP by all test systems (all p<0.001). A rather poor correlation was observed between AA-induced platelet reactivity by LTA and the VerifyNow aspirin assay (r = 0.15, p = 0.007), while both methods did not correlate with MEA. AA-induced platelet reactivity by all aggregation tests correlated significantly, but rather poorly with AA-induced P-selectin expression (all p<0.05), while only AA-induced platelet reactivity by LTA correlated significantly with AA-induced activated GPIIb/IIIa (r = 0.21, p<0.001). The platelet surface expression of P-selectin in response to AA was significantly higher in patients with HRPR by LTA AA and MEA AA (both p<0.02). In contrast, P-selectin expression in response to AA was similar in patients without and with HRPR by the VerifyNow aspirin assay (p = 0.5), and platelet surface activated GPIIb/IIIa in response to AA did not differ significantly between patients without and with HRPR to AA by all test systems (all p>0.1). In conclusion, ADP-induced platelet reactivity by aggregometry translates partly into flow cytometry. In contrast, AA-induced platelet reactivity correlates poorly between different platelet aggregation tests, and between aggregometry and flow cytometry. Overall, both approaches capture different aspects of platelet function and are therefore not interchangeable in the assessment of agonists´-induced platelet reactivity. Clinical outcome data are needed to determine which test systems and settings are associated with different in vivo consequences.     

Pneumococcal polysaccharide vaccination in adults undergoing immunosuppressive treatment for inflammatory diseases – a longitudinal study

IntroductionPatients undergoing immunosuppressive therapy are at increased risk of infection. Community-acquired pneumonia and invasive pneumococcal disease account for substantial morbidity and mortality in this population and may be prevented by vaccination. Ideally, immunization to pneumococcal antigens should take place before the start of immunosuppressive treatment. Often, however, the treatment cannot be delayed. Little is known about the efficacy of pneumococcal vaccines during immunosuppressive treatment. The objectives of this study were to determine the percentage of vaccine-naïve, immunosuppressed adults with inflammatory diseases seroprotected against Streptococcus pneumoniae and to assess factors associated with the immunogenicity, clinical impact and safety of 23-valent pneumococcal polysaccharide vaccine (PPV) in seronegative subjects.MethodsThis observational study included patients 18 years of age and older who were receiving prednisone ≥20 mg/day or other immunosuppressive drugs. Exclusion criteria were PPV administration in the previous 5 years, intravenous immunoglobulins and pregnancy. Serum immunoglobulin G (IgG) antibody levels against six pneumococcal serotypes were measured. Seropositivity was defined as IgG of 0.5 μg/ml or greater for at least four of six serotypes. Seronegative patients received PPV, and seropositive patients were included as a comparison group. Vaccine response and tolerance were assessed after 4–8 weeks. Disease activity was evaluated on the basis of the Physician Global Assessment scores. Serology was repeated after 1 year, and information on any kind of infection needing medical attention was collected. Outcomes were the proportion of seropositivity and infections between vaccinated and unvaccinated patients.ResultsOf 201 included patients, 35 received high-dose corticosteroids and 181 were given immunosuppressive drugs. Baseline seronegativity in 60 (30 %) patients was associated with corticotherapy and lower total IgG. After PPV, disease activity remained unchanged or decreased in 81 % of patients, and 87 % became seropositive. After 1 year, 67 % of vaccinated compared with 90 % of observed patients were seropositive (p < 0.001), whereas the rate of infections did not differ between groups. Those still taking prednisone ≥10 mg/day tended to have poorer serological responses and had significantly more infections.ConclusionsPPV was safe and moderately effective based on serological response. Seropositivity to pneumococcal antigens significantly reduced the risk of infections. Sustained high-dose corticosteroids were associated with poor vaccine response and more infections.     

High-dose IgG for post-transfusion purpura-revisited

Therapy for post-transfusion purpura (PTP) is controversial. We have evaluated the effect of high-dose IgG (HDIgG) in 11 PTP cases investigated in our institution and summarized the clinical data of 8 additional cases reported in the literature. Two of these 19 cases had to be eliminated from the analysis (1 patient received a total dose of less than 30 g of IgG; 1 patient died 2 days after starting HDIgG therapy from congestive heart failure). Out of a total of 17 cases, 16 had good or excellent response reaching normal platelet values within few days; only one failure was observed. Five patients relapsed, but attained complete remission after a second course (dose) of IgG. Total doses per course ranged between 52 and 180 g of IgG. Five different IgG preparations were used and seemed similarly effective. No adverse reactions were observed. We conclude that HDIgG is the treatment of choice for PTP.     

Age-related changes in in vitro immunoglobulin secretion: Comparison of responses to T-dependent and T-independent polyclonal activators

In vitro Ig secretion by peripheral blood mononuclear cells (MNCs) from old and young donors, in response to T-dependent (TD) [pokeweed mitogen (PWM)] and T-independent (TI) [Salmonella paratyphii B (SPB)] activation were compared. In older donors, the IgG and IgA responses to PWM were comparable to those of young donors; the IgM response was reduced in the elderly. With SPB activation, IgA response was again preserved, whereas IgG response was reduced and IgM secretion was markedly decreased. These data indicate class-specific changes in Ig responsiveness to both TD and TI cell activators with age. The reduction in TI-induced IgG and IgM responses in the elderly suggest that changes in B cells themselves have occurred. The preservation of the TD IgG response in concert with reduced TI response indicates that a decline in T-suppressor influences over B cells in the elderly coupled with reduced B-cell synthesizing capacity can result in apparent “preservation” of the final Ig response. In keeping with the above postulate, analysis of individual elderly donors' responses indicated that some of the old donors responded to PWM, but not SPB; none of the old donors responded to SPB and not PWM. In contrast, some young donors did respond to SPB, but not PWM. These results also suggest that nonresponse to PWM in young donors relates to an override of functionally intact B cells by T-regulator influences.     

Thrombospondin-bound integrin-associated protein (CD47) physically and functionally modifies integrin alphaIIbbeta3 by its extracellular domain

Integrin-associated protein (IAP/CD47) is a receptor for the C-terminal cell binding domain of thrombospondin (TS). A peptide from the C-terminal cell binding domain, KRFYVVMWKK (4N1K) binds to IAP and stimulates the integrin-dependent cell functions, including platelet aggregation. We investigated the mechanism by which TS-bound IAP modulates the affinity of platelet integrin, alphaIIbbeta3. Platelet aggregation induced by 4N1K was not completely inhibited by energy depletion with sodium azide and 2-deoxy-d-glucose, although ADP or collagen-induced platelet response was completely inhibited. The binding of ligand-mimetic antibody PAC1 to alphaIIbbeta3 was also induced in the energy-depleted platelets. In the transfected Namalwa cells, 4N1K induced activation of the alphaIIbbeta3 with mutated beta3 (Ser-752 to Pro), which is a non-responsive form to inside-out signaling, as well as wild type alphaIIbbeta3. The truncated form of IAP with only the extracellular immunoglobulin-like (Ig) domain was sufficient for the activation of alphaIIbbeta3 in Chinese hamster ovary cells, although the IAP-mediated intracellular signaling was abolished, which was monitored by the absence of down-regulation of mitogen-activated protein kinase phosphorylation. Furthermore, the soluble recombinant Ig domain of IAP induced PAC1 binding to alphaIIbbeta3 on Chinese hamster ovary cells when added with 4N1K. Physical association between the soluble recombinant Ig domain of IAP and purified alphaIIbbeta3 was detected in the presence of 4N1K. These data indicate that the extracellular Ig domain of IAP, when bound to TS, interacts with alphaIIbbeta3 and can change alphaIIbbeta3 in a high affinity state without the requirement of intracellular signaling. This extracellular event would be a novel mechanism of affinity modulation of integrin.     

Stem cell transplantation for metastatic breast cancer: analysis of tumor contamination

The purpose of this study was to determine the efficacy, engraftment kinetics, effect of bone marrow tumor contamination, and safety of high-dose therapy and granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood progenitor cell (PBPC) support for patients with responding metastatic breast cancer. Forty two patients underwent G-CSF (10 μg/kg) stimulated PBPC harvest. PBPC and bone marrow aspirates were analyzed by histologic and immunocytochemical methods for tumor contamination. Thirty-seven patients received high-dose therapy consisting of cyclophosphamide 6 g/m², thiotepa 500 mg/m², and carboplatin 800 mg/m² (CTCb) given as an infusion over 4 d followed by PBPC reinfusion and G-CSF (5 μg/kg) support. No transplant related deaths or grade 4 toxicity was recorded. CD34+ cells/kg infused was predictive of neutrophil and platelet recovery. With a median follow-up of 38 months, three year survival was 44% with relapse-free survival of 19%. Histological bone marrow involvement, found in 10 patients, was a negative prognostic factor and was associated with a median relapse-free survival of 3.5 months. Tumor contamination of PBPC by immunohistochemical staining was present in 22.5% of patients and found not to be correlated with decreased survival. G-CSF stimulated PBPC collection followed by a single course of high dose chemotherapy and stem cell infusion with G-CSF stimulated marrow recovery leads to rapid, reliable engraftment with low toxicity and promising outcome in women with responding metastatic breast cancer.