X-linked thrombocytopenia in a female with a complex familial pattern of X-chromosome inactivation.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, eczema and various degrees of immune deficiency caused by mutations in the WAS gene, which encodes the WASP protein, the expression of which is restricted to haematopoietic cells. Mild allelic variants are associated with X-linked thrombocytopenia (XLT). Female carriers tend in general to be asymptomatic as a consequence of a positive selection of cells with an active normal X chromosome, which results in a non-random inactivation of the mutated gene in affected cell lineages. We report on six female members of the same family carrying the mutated WAS allele p.V332A, which is known to be associated with XLT. One of them had presented severe thrombocytopenia from birth. Western blotting showed the WASP protein in peripheral blood cells to be normal in size and expression, and scanning electron microscopy revealed a normal distribution of microvilli on T cells. X-chromosome inactivation-pattern analysis showed total inactivation of the non-mutated paternal X chromosome in the patient's peripheral blood cells. All the other female family members were healthy and presented varying X-chromosome inactivation patterns, ranging from random X chromosome inactivation to total X-chromosome inactivation of the mutated chromosome. Our results in these female carriers of p.V332A show that manifestation of the disease requires a total inactivation of the non-mutated X chromosome and allow us to confirm that clinical manifestations in female carriers are highly dependent not only on the mutation characteristics but also on the X-chromosome inactivation pattern of affected line.

CD27+ B cells from a subgroup of common variable immunodeficiency patients are less sensitive to apoptosis rescue regardless of interleukin-21 signalling.

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinaemia and recurrent infections. Although the underlying cause is unknown, B cells from most CVID patients fail to differentiate to memory or plasma cells. We investigated if increased apoptosis could influence the fate of B cells. For this purpose we activated purified B lymphocytes of CVID patients with a surrogate T-dependent (anti-CD40) or T-independent [cytosine-phosphate-guanosine oligodeoxynucleotides (CpG-ODN) or anti-immunoglobulin (Ig)M)] stimulus with or without interleukin (IL)-21. We found that CD27(+) B cells were more sensitive than CD27(-) B cells to spontaneous apoptosis and less sensitive to rescue from apoptosis. The addition of IL-21 down-modulated the protective effect of all the stimuli on CD27(-) B cells and the protective effect of CpG-ODN and anti-IgM on CD27(+) B cells. In contrast, IL-21 rescued unstimulated CD27(-) B cells and improved the rescue of anti-CD40-stimulated CD27(+) B cells. When we compared patients and controls, mainly CD27(+) B cells from MB0 patients were less sensitive to rescue from apoptosis than those from MB1 patients and controls after activation, irrespective of the IL-21 effect. Increased apoptosis during an immune response could result in lower levels of immunoglobulin production in these patients.

Advancing allele group-specific amplification of the complete HLA-C gene--isolation of novel alleles from three allele groups (C*04, C*07 and C*08).

A variety of strategies have been designed for sequence-based HLA typing (SBT) and for the isolation of new human leucocyte antigen (HLA) alleles, but unambiguous characterization of complete genomic sequences remains a challenge. We recently reported a simple method for the group-specific amplification (GSA) and sequencing of a full-length C*04 genomic sequence in isolation from the accompanying allele. Here we build on this strategy and present homologous methods that enable the isolation of HLA-C alleles belonging to another two allele groups. Using this approach, which can be applied to sequence-based typing in some clinical settings, we have successfully characterized three novel HLA-C alleles (C*04:128, C*07:01:01:02, and C*08:62).

Specific IgE levels to Dermatophagoides pteronyssinus are associated with meteorological factors.

BACKGROUND: Dermatophagoides pteronyssinus specific IgE (sIgE) measurement is a major diagnostic test for the detection of sensitization to that allergen. METHODS: To investigate the effect of climate on the seasonal variations of D.pteronyssinus sIgE, we studied the tests performed in an insular population during a 10-year period. The association with meteorological factors was evaluated with multiple regression analyses. RESULTS: Of 24,879 tests performed for D. pteronyssinus sIgE, 16,719 (67.2%) were D. pteronyssinus sIgE positive; 24.5% were tested for asthma and 46.07% for rhinitis. D. pteronyssinus sIgE levels showed a seasonal pattern with an annual peak in November. In the multivariate analyses solar radiation (r = -0.94) and relative humidity (r = 0.86) were independent factors associated with D. pteronyssinus sIgE levels. The resulting model could explain 93% (p < 0.001) of D. pteronyssinus sIgE variability. CONCLUSIONS: Our population showed a seasonal pattern of D. pteronyssinus sIgE explained by relative humidity and solar radiation.

Neonatal mixed lineage acute leukaemia.

We report here an uncommon case of neonatal acute leukaemia that presented concomitant with serological evidence of rubella infection. The clinical course was aggressive and the patient died 5 days after diagnosis from septicaemia. Leukaemic blasts had a mixed lineage immunophenotype co-expressing a constellation of B-lymphoid (CD19, cytCD22, TdT) and myeloid (CD13, CD33, CD14, anti-MPO) markers, as well as multiple adhesion molecules and markers associated with early lympho-myeloid progenitor cells (CD34, CD7, HLA-DR). A previously unrecorded discordant expression of different CD10 and CD34 epitopes was identified using different monoclonal antibodies. The karyotype was 46,XX t(4;11)(q21;q23) and molecular analysis confirmed rearrangement of the trithorax-related oncogene HRX at 11q23. There was a clonal biallelic rearrangement of the immunoglobulin heavy-chain gene. The features of this rare case have implications for possible aetiological events leading to leukaemia.

Pre-B Acute Lymphoblastic Leukemia During a Complete Remission in T-Acute Lymphoblastic Leukemia.

This case provides evidence for a phenotypic switch from a characteristic T-ALL into a typical pre-B ALL. A 17 years old boy presented with massive hepatosplenomegaly and a mediastinal mass. The blasts were characterized as L2 type according to the French, American and British (FAB) classification and the staining with acid phosphatase was positive. The immunophenotype was CD2 +, CD5 +, CD7 + and CD10 -. The patient achieved a complete remission but relapsed 4 years later. At relapse, a striking immunological shift was apparent. The blast cells were now morphologically LI subtype and displayed the CD10 +. C{mew} + phenotype but were CD2 -, CD5 -, CD7 -. These findings are consistent with a transformation of the initial clone although the development of a second leukemia in the same patient could not be excluded with complete certainty.

A simplified method for cryopreservation of hematopoietic stem cells with -80 degrees C mechanical freezer with dimethyl sulfoxide as the sole cryoprotectant.

A simplified method for cryopreservation was developed with 10% dimethylsulfoxide (DMSO) as the sole cryoprotectant without rate-controlled freezing. This method produced high recovery rate for mononucleated cells (87%) and elevated trypan blue viability (90%). Autologous peripheral blood stem cells (PBSCs) and bone marrow cells with plasma and 10% DMSO were frozen and stored in a -80 degrees C mechanical freezer. Eleven patients with solid and hematological malignancies were transplanted with autologous bone marrow or PBSCs. The median number of infused mononuclear cells (MNC) and CD34+ cells were 3.63 x 10(8)/Kg and 4.80 x 10(6)/Kg, respectively. The median number of infused post-thawing CFU-GM was 20 x 10(4)/Kg. All patients showed a rapid and sustained engraftment. The mean times to reach a neutrophil count of 0.5 x 10(9)/L and a platelet count of 50 x 10(9)/L were 11 and 13 days, respectively. All patients are alive and 10 in unmaintained complete remission for 3-9 months after transplantation. These results show the efficacy of this simplified cryopreservation technique that will be useful for institutions without rate-controlled freezing facilities.

A functional vagotomy induced by unilateral forced right nostril breathing decreases intraocular pressure in open and closed angle glaucoma.

There is evidence of the central regulation of intraocular pressure, and it has been suggested that vagal tone might be increased in glaucoma simplex. The nasal cycle, the simultaneous congestion-decongestion response in the nasal cavities, reflects the dynamic lateralisation of the autonomic nervous system. Since this lateralisation presents with sympathetic activity induced by left brain hemisphere stimulation and parasympathetic activity induced by right hemisphere stimulation, it was subsequently demonstrated that forced unilateral nostril breathing induces selective contralateral hemispheric stimulation as measured by relative increases in the electroencephalographic amplitude in the contralateral hemisphere as well as alternating lateralisation of plasma catecholamines. Using this functional vagotomy, we report that left hemispheric stimulation by 20 minutes of forced unilateral right nostril breathing led to a significant bilateral decrease of 4.6 mmHg (25%) in intraocular pressure in 46 patients with open and closed angle glaucoma. However, it significantly increased the IOP in three patients with neovascular, one with juvenile onset, and one with closed angle glaucoma.

Treatment of vernal keratoconjunctivitis a double-masked study.

Thirty-one children aged five to 14 years with severe vernal keratoconjunctivitis (VKC) were enrolled in a double-masked study evaluating the efficacy of three ophthalmic preparations: cromolyn sodium (Opticrom) 2%, artificial tears and fluorometholone 0.1% (FML). These drugs were coded respectively as A, B, C, and dispensed in similar bottles. Both eyes were treated, and drug assignment was made at random. Objective assessment of the disease activity was done under slit lamp by two ophthalmologists according to a five-point scale. The results were tabulated and the chi-square test performed. Decoding of the drugs was done only after completion of the statistical analysis. FML was found effective with a statistically significant difference from the other two drugs, both after the first week on the initially assigned treatment (p = 0.05) and on completion of the treatment period according to protocol (p = 0.005). Although several patients improved under cromolyn sodium, there was no statistically significant difference between cromolyn sodium 2% and artificial tears. All the preparations were well tolerated. One patient developed bilateral allergic blepharitis when treated with FML during the study. Another patient who responded well to FML developed posterior subcapsular cataract while continuing treatment with corticosteroids a few months after completion of the study.

Treatment of severe vernal keratoconjunctivitis with cyclosporine A eyedrops.

Twenty-one children (4 to 12 years old) with severe vernal keratoconjunctivitis refractive to treatment with corticosteroid eyedrops and/or 2% disodium cromoglycate (Optic-rom, Fisons) were treated with CsA 2% eyedrops in oil solution. Eighteen patients (86%) experienced a rapid relief of their subjective symptoms three to four days after initiation of treatment. This was accompanied by objective improvement of ocular manifestations and visual acuity within a week. Sixteen of the children (76%) remained controlled during the 6-week period of treatment. However, only five patients (24%) did not need any additional therapy 2 months after discontinuation of the CsA eyedrops. These findings can be interpreted as an indication for the possible involvement of interleukin secretion in the clinical manifestations of vernal keratoconjunctivitis. Alternatively, the beneficial effects of CsA in this disease may be due to a direct effect on the mast cells preventing the release of their mediators.

Treatment of endogenous uveitis with cyclosporine A.

Thirty patients suffering from severe sight-threatening bilateral chronic endogenous uveitis were treated with initial dosages of 10 mg/kg/d of CsA. After 1 month of therapy, all patients but one demonstrated a rapid decrease of the intraocular inflammatory processes along with an arrest of the deterioration of vision. After 1 year (or more) of treatment, most patients still show the same visual acuity achieved after 1 month. During the 3-year span of this study, attempts at tapering off the CsA dosage to less than 5 mg/kg/d induced a temporary flare-up in 20 of the 24 patients followed without interruption for more than 6 months. Nonetheless, control of the intraocular inflammation was finally achieved by 5 mg CsA/kg/d or less in 14 of the 25 patients. In six of these 14 patients the CsA dosage was further tapered to total discontinuation. Two of the six patients showed a rapid reactivation of the intraocular inflammatory processes with a profound decrease in vision within 1 and 3 weeks, respectively. In four patients, control of the intraocular inflammation and preservation of good visual acuity have been observed for a period of up to 18 months.