High-titre factor VIII inhibitor in two children with mild haemophilia A.

A frequently encountered complication of therapy given to patients with severe haemophilia A is the development of antibodies to infused factor VIII. While much less common, inhibitors also occur in patients with mild or moderate severity haemophilia A. Often thought to be of low titre and transient, several cases of high-titre inhibitors have been described in patients with mild or moderate haemophilia A. Generally these occur in adults or adolescents following significant infused factor VIII exposure. A review of reported cases revealed only two cases of high-titre inhibitor formation in mild haemophilia A patients younger than 10 years of age. We wish to report our experience with an additional two children with mild haemophilia A and high titre inhibitors, and offer suggestions for the management of these children.

Inhibition of thymopoiesis of CD34+ cell maturation by HIV-1 in an in vitro CD34+ cell and thymic epithelial organ culture model.

The mechanisms by which HIV-1 affects thymopoiesis were determined by preincubating CD34+ cells or cultured thymic epithelial (CTE) cells with lymphotropic (T-) and monotropic (M-) strains of HIV-1 in an in vitro CTE organ and CD34+ cell coculture model that allows for analysis of development of thymocytes and mature T cells. When purified CD34+ cells were precultured with either T- or M-tropic strains of HIV-1, thymopoiesis was impaired in a two-week coculture manifested by decreased cell number of thymocytes generated. However, the percentages of thymocyte subpopulations were comparable to control uninfected cocultures. Furthermore, HIV infection of thymocytes was predominantly observed in the CD44+CD3- population. However, in a four-week coculture experiment, HIV infection and depletion of more mature thymocytes were also observed. When CTE cells were preincubated with T- and M-tropic strains of HIV before addition of CD34+ cells, the number of thymocytes and subpopulations of thymocytes at early and later stages of maturation were markedly decreased. Furthermore, CD34+ and CD44+CD3- cells become HIV-infected. In summary, HIV-1 infection inhibited thymocyte maturation at early stages of thymocyte maturation CD44+CD25-CD3-. In addition, HIV also depleted later stages of CD4+ thymocyte subpopulations.

Thymosin-alpha1 stimulates maturation of CD34+ stem cells into CD3+4+ cells in an in vitro thymic epithelia organ coculture model.

The effect of thymosin-alpha1 on thymopoiesis is largely unknown. Thymosin is found in the cortical and medullary thymic epithelia, as well as in nurse cells; thus, it is hypothesized that thymosin may affect both early and late stage of thymocyte maturation. In this study, the effect of thymosin-alpha1 on thymopoiesis was determined by coculturing in vitro CD34+ stem cells (SC) with allogeneic cultured thymic epithelia fragments (CTEF) for 1-4 weeks and analyzing T-cell maturation by flow cytometry. Thymosin-alpha1 significantly enhanced the cell number (e.g., proliferation) of mononuclear cells obtained at 2 and 4 weeks of the SC-CTEF cocultures (P < 0.01 and < 0.05, respectively). In particular, thymosin-alpha1 stimulated expression of CD3+ cells at 3 and 4 weeks (P < 0.05). The predominant subpopulation increased by thymosin stimulation was single positive mature CD4+ cells, which was confirmed to occur within the SC-CTEF thymic organ tissue by laser confocal immunofluorescence microscopy. Thymosin stimulation tended to enhance IL-7 synthesis, critical cytokine in the maturation of thymocytes. In summary, this is the first study to demonstrate that thymosin-alpha1 enhanced thymopoiesis of CD34+ stem cells in humans using an in vitro model of differentiation using stem cells and cultured thymic epithelial fragments cocultures. Furthermore, the thymosin significantly increased expression of CD3+4+ T cells.

Development of a method of thymocyte differentiation of bone marrow-enriched CD34+CD38- cells in postnatal allogeneic cultured thymic epithelia to evaluate immunodeficiency disorders.

An in vitro model of CD34+CD38- stem cell (SC) differentiation in postnatal cultured thymic epithelia fragment (CTEF) cocultures is described. Sequential phenotypic analysis of the progeny of the SC-CTEF demonstrated predominantly thymocytes and minor populations of promyelocytes, monocytes and natural killer cells. Triple-positive CD3+CD4+CD8+, double-positive CD4+CD8+, and mature single-positive CD4+ and CD8+ T cells, which were TCR alpha beta+, were identified indicating normal thymocyte maturation. In kinetic studies, mature single-positive CD4+ T cells increased from 29% of total cells at one week to 54% at four weeks of coculture. These findings demonstrate that coculture of bone marrow-derived SC and allogeneic cultured thymic epithelia in vitro results in continuous normal predominantly thymocyte differentiation. The SC-CTEF cocultures were then infected with two different strains of human immunodeficiency virus. CD4+ thymocytes were markedly decreased. However, inhibition of early thymocyte maturation steps was also suggested by the presence of increased triple-negative and CD44+CD25-CD3-thymocytes and decreased CD44+CD25+ thymocytes. This model system of thymocyte maturation will be useful in the evaluation of primary T cell immunodeficiency disorders, gene therapy of SC and pharmacological augmentation of thymic function.

Arrest of in vitro T cell differentiation of normal bone marrow-derived CD34+ stem cells with thymic epithelial fragments from children with AIDS.

A novel approach is presented to assess the ability of thymic tissues obtained from children with end stage AIDS to attract normal bone marrow (BM)-derived CD34+ (lineage negative) stem cells (SCs) and support lymphopoiesis in vitro. Chemokinesis of BM-derived CD34+ SCs was analyzed by time-lapse videomicroscopy to ascertain whether an alteration in SC motility could contribute to abnormal thymopoiesis under conditions of HIV infection. The migration of SCs derived from an HIV+ donor into thymic tissue was not significantly altered compared to normal controls, as were normal SCs migrating toward thymic epithelial cell monolayers derived from an HIV+ patient. Thymic tissue obtained from children with AIDS contained nests of CD34+ SCs identified by immunofluorescence, indicating SC homing to the thymus is apparently supported in HIV infection. The ability of HIV-affected thymic epithelial fragments to support lymphopoiesis was determined by examining the initial thymocyte populations present, compared to thymocytes produced de novo in T cell-depleted thymic fragments, following a single pulse of lineage negative CD34+ CD38- SCs. In comparison to normal controls, thymocytes derived from the HIV-affected thymic epithelial fragment coculture had an increased percentage of triple negative thymocytes (28% of lymphocytes from HIV-affected tissue versus 1.5% in controls, p < 0.01) and a decreased percentage of double and single positive CD4+ thymocytes. However, CD3+CD8+ TCR alpha beta + expression was comparable to control cultured thymic epithelial fragments indicating that HIV-affected thymic epithelia were capable of supporting the development of the CD8+ lineage. In an effort to extend the information obtained to date from the histological examination of HIV-affected thymic tissue, select patient thymic tissues were maintained in culture to evaluate the capacity of undifferentiated thymic epithelial cell guirlandes to differentiate in vitro. A partial regeneration of certain subpopulations of the thymic epithelium defined by TE-4 monoclonal antibody (mAb) and CDR2 mAbs occurred during the in vitro culture. The epithelial and mesenchymal components of thymic tissues were distinguished by immunostaining for keratins (indicative of epithelium) and vimentin (a mesenchymal marker). Further evaluation of the modulation of HIV thymus, with respect to the testing of new therapeutic strategies on SCs, will be possible with this in vitro model.

Abnormal in vitro thymocyte differentiation in a patient with severe combined immunodeficiency-Nezelof's syndrome.

An in vitro coculture model system of CD34+ stem cells and allogenic cultured thymic epithelia fragments was used to evaluate thymocyte differentiation in a 9-month-old child of Amish descent with Nezelof syndrome. Though the patient's stem cells differentiate to acquire normal expression of CD2 and CD7, later steps of maturation were abnormal. There was detectable but reduced expression of CD3 and CD4 phenotypes. CD44+ expression, however, was markedly reduced. CD44 is an adhesion molecule, interacting with the matrix ligands hyaluronan and fibronectin, and is expressed early in thymocyte differentiation and subsequently in mature T cells. It is hypothesized that abnormal expression of CD44 in a variant of severe combined immunodeficiency, Nezelof's syndrome, interferes with normal thymocyte and thymic epithelial interaction, which leads to abnormal thymocyte differentiation.

T cell differentiation/maturation of CD34+ stem cells from HIV-seropositive hemophiliacs in cultured thymic epithelial fragments.

The clinical manifestations of AIDS are predominantly due to the cellular and humoral immune dysfunction caused by HIV infection, and thymic dysplasia caused by HIV infection probably contributes to the T cell lymphopenia. In the present study, T cell differentiation and/or maturation was assessed when enriched CD34+ stem cells (SCs or SC) purified from bone marrow of HIV-seropositive hemophiliacs were cocultured with allogeneic cultured thymic epithelial fragments (CTEFs). When HIV-seropositive hemophiliacs' enriched CD34+ SC were cocultured with allogeneic CTEFs, acquisition of the T cell phenotypic markers CD7, CD2, CD3, CD4, CD8 and T cell receptor for antigen (TCR) alpha beta was observed from cells harvested from the culture media peaking at approximately 28 days. Origin of the differentiated and matured T cells from the CD34+ SC was confirmed by labeling the SC with 5-(and -6)-(((4-chloromethyl)benzoyl)amino)tetra-methyl-rhodamine (CMTMR), a fluorescent cytoplasmic dye, and detecting fluorescence in the differentiated and matured T cell by flow cytometry. In one experiment, CMTMR labeling was omitted and double positive CD4+CD8+ and triple positive CD3+CD4+CD8+ thymocytes were identified. These studies confirmed that thymocyte differentiation/maturation from SC had occurred. In addition, T cells obtained from the CD34+ SC and CTEF cocultures proliferated to phytohemagglutinin stimulation maximally with stem cell donor antigen-presenting cells (APCs) and also proliferated to pooled B cells in a mixed lymphocyte culture (MLC). Furthermore, the T cells produced were tolerant to thymus donor B cell HLA antigens (p < 0.025); though there was slight MLC reactivity to autologous stem cell donor B cell HLA compared to thymic B cells (p < 0.025). These T cells demonstrated positive self-alloreactivity to stem cell HLA antigens in four of nine persons, though decreased compared to pool B cell alloantigens. Furthermore, in three experiments, responsiveness to stem cell donor B cells subsequently disappeared upon further duration of CD34+ SC-CTEF coculture. These studies suggested that CD34+ SC gave rise to accessory cells populating the thymus that contributed to HLA restriction. To further evaluate this hypothesis, two different donors of CD34+ SC were cultured simultaneously with thymic epithelial fragments and MLC reactivity was then examined toward APC of the stem cell donors. In these experiments, T cells responded to stimulation with HLA antigens of the pool B cells and did not respond to thymus donor B cells. In six of eight experiments, the chimeric SC-CTEF T cells did not respond to stimulation with B cells of either stem cell donor. These studies suggest that HLA restriction and tolerance were induced by cells of the stem cell donor as well as the thymic epithelial cell HLA antigens. In summary, these studies demonstrated that HIV-infected hemophiliac bone marrow-derived nonadherent CD34+ SC were capable of differentiating and/or maturing into T cells when cocultured in a normal allogeneic thymic environment. Furthermore, the T cells derived from derived CD34+ SC were capable of differentiating into T cells when cocultured in a normal allogeneic thymic environment, proliferated maximally with APCs from the stem cell donor and were tolerant of thymic HLA class II antigens, and to a lesser degree to stem cell donor B cell HLA antigens.

Decrease of CD4 cell number and function in HIV-seropositive hemophiliacs in a longitudinal study.

The Regional Hemophilia Center in St. Louis initiated a prospective study beginning in 1982 to measure sequentially T-cell subpopulations and in vitro lymphoproliferative responses in hemophilia A patients. In a cohort of 106 hemophiliacs, the prevalence of HIV-seropositivity increased from 46.7% in 1982 to 74.5% by 1987. There was a persistent gradual decline over time of T helper/inducer (CD4) cells in HIV-seropositive hemophiliacs (P less than .01). This was reflected by an increasing percentage of hemophiliacs with abnormally low CD4 cells (less than 2 standard deviations below the mean of normal individuals) from 6.7% in 1983 to 52.4% in 1987. Function of CD4 cells, as estimated by in vitro lymphoproliferative responses to phytohemagglutinin (PHA) and tetanus toxoid stimulations also demonstrated a decline over the same years. Lymphoproliferative responses to PHA by HIV-seropositive hemophiliacs' mononuclear cells (MNC) declined from a 90.2% normal response in 1983 to a 71.7% normal response in 1987 (P less than .05). Decreased responses to stimulation with the soluble antigen tetanus toxoid were also seen from 1983 compared with 1987 (P less than .05). This was due to an increased percentage of HIV-seropositive hemophiliacs' MNC, which were unresponsive to stimulation to tetanus toxoid (stimulation index less than 3.0) from 20.8% in 1983 to 41.0% in 1987. These findings indicate that HIV-infection was associated over time with a decline of CD4 number and function in a substantial portion of hemophiliacs.

HIV and hemophilic children's growth.

The acquired immune deficiency syndrome (AIDS) often has profound effects on growth; however, the effects of human immunodeficiency virus (HIV) on asymptomatic children's growth are unknown. Before heat inactivation/HIV donor screening of factor concentrates, many hemophilic children became infected with HIV. We evaluated four hemophilic groups without AIDS, using age-standardized growth parameters: group 1, 41 HIV-seropositive children (median age of 13 years); group 2, 11 HIV-seronegative children (median age of 4 years); group 3, 20 children frequently receiving concentrates, evaluated before 1979 (median age of 9 years); and group 4, 11 children rarely receiving concentrates, evaluated before 1979 (median age of 6 years). Median height for age (HA), weight for age (WA), and weight for height (WH) of groups 1 and 2 exceeded the 50th percentile of referent norms. HA, WA, WH, and weight/height did not vary significantly by group, nor did these decline over periods of 11 to 70 months. However, for those less than 11 years of age in group 1, HA declined by 25 percentile points over at least a 3 year period. Also, group 1's T helper-to-suppressor cell ratios at 12 +/- 3 months following the latest growth evaluation were positively associated with both HA and WA at the last evaluation. Eight children were evaluated before 1979 and again after they seroconverted to HIV.(ABSTRACT TRUNCATED AT 250 WORDS)

Conservative management of spinal epidural hematoma in hemophilia.

Two patients with hemophilia and spinal epidural hematoma, who were treated successfully with serial Factor VIII infusions, are reported. This form of conservative therapy may circumvent the need for decompressive laminectomy and its attendant complications in instances in which the neurologic deficit is mild or stable. Somatosensory evoked potential studies were useful in documenting spinal cord dysfunction in 1 of the 2 patients.

Time relationship of immune changes to HTLV-III/LAV seroconversion in patients with hemophilia.

Longitudinal immune studies of patients with hemophilia A were begun in 1982 by the Regional Hemophilia Center in St. Louis, Missouri. Serum samples collected from 74 participants between 1982 and 1985 were analyzed for antibody to human T-lymphotropic virus type III (HTLV-III)/lymphadenopathy-associated virus (LAV). The incidence of antibody to HTLV-III/LAV has increased significantly in this population of patients with hemophilia. Only one of eight hemophiliacs had detectable antibody before July 1982, whereas 88.7% (55/62) were positive in 1985. T-cell surface markers were markedly abnormal in seropositive hemophilia patients with decreased percentage and number of OKT4-positive cells compared with seronegative hemophiliacs and controls. Lymphoproliferative responses to mitogens and antigens were normal in seronegative hemophilia patients. Seropositive hemophiliacs, compared with seronegative hemophiliacs, had significantly decreased lymphoproliferative responses, especially to pokeweed mitogen, tetanus, and Candida stimulations. Immune studies of seven HTLV-III/LAV seropositive hemophiliacs revealed antigen unresponsiveness and decreased T4 cells 2 to 32 months prior to development of full-blown AIDS. Longitudinal immune studies from 1983-85 revealed increasing number of seropositive hemophiliacs with antigen unresponsiveness and decreased T4 cells.

HTLV-III/LAV antibody status of spouses and household contacts assisting in home infusion of hemophilia patients.

Thirty-four adult and pediatric hemophilia A and B patients and 50 nonhemophilic members belonging to 28 families were enrolled in August 1984 in a study of human T cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) antibody status and T cell subpopulation numbers. All 50 household contacts, including three spouses of LAV antibody-positive adult hemophiliacs, were immunologically normal and serologically negative with respect to HTLV-III/LAV. Based on Western blot serologic testing of blood samples collected intermittently between July 1981 and August 1984 from 33 representative St Louis hemophiliacs studied during the period from 1981 to 1984, the average time since seroconversion was estimated as 20 months. One spouse of a seropositive hemophiliac and 23 parents of 27 seropositive pediatric hemophiliacs assisted regularly with home infusions. These infusion assistants have collectively experienced 44 person-years of concentrate infusion "exposure" without seroconversion. These results suggest that the likelihood for transmission of HTLV-III/LAV from hemophiliacs to persons assisting in their therapy is extremely low.

Chronic idiopathic thrombocytopenic purpura followed by chronic myelocytic leukemia.

An 8-year old girl was diagnosed as having idiopathic thrombocytopenic purpura which later became refractory to steroids, splenectomy, and immunosuppressive therapy. While she was being treated for pneumococcal meningitis, 7 1/2 years later, she was found to have Philadelphia-chromosome-positive chronic myelocytic leukemia. She died 2 1/2 years after the diagnosis of leukemia. The association of autoimmune disorders and hematologic malignancies has been well-recognized. To our knowledge, there has been no previous report of chronic idiopathic thrombocytopenic purpura preceding the development of chronic myelocytic leukemia in the literature.

Aortic valve replacement in von Willebrand's disease.

A patient with von Willebrand's disease having aortic valve replacement was managed with cryoprecipitate infusions and monitoring of factor VIII levels. This disorder is associated with low factor VIII levels and abnormal platelet function. There may be no history of bleeding, as the severity of the bleeding tendency varies greatly and fluctuates temporally. The partial thromboplastin time is frequently prolonged, but more detailed studies are necessary to establish a diagnosis (bleeding time, platelet adhesiveness to glass beads and ristocetin, von Willebrand's antigen, ristocetin-von Willebrand's factor, and factor VIII clotting activity). Elevation of factor VIII levels to 50 to 100% of normal allows adequate clotting and is best accomplished with cryoprecipitate or fresh frozen plasma rather than commercial concentrates of factor VIII, whose activity is unpredictable.

Classic hemophilia A in a female.

A 53-year-old woman with a history of recurrent bleeding complications since childhood and a positive family bleeding history, previously diagnosed as von Willebrand's disease, was investigated. She was found to have a markedly reduced level of antihemophilic factor (AHF) activity, a low AHF activity/AHF antigen ratio, normal Ristocetin-induced platelet aggregation and a normal level of von Willebrand factor activity. These findings were consistent with the diagnosis of classic hemophilia A which was confirmed by the results of similar studies in nine of the patient's relatives. The report illustrates the usefulness of newer laboratory methods in the differentiation between classic hemophilia A and von Willebrand's disease which may have important clinical implications.