Lymphocyte subpopulations do not alter during blood storage at 4 degrees C

Recent articles have suggested that lymphocyte subpopulations, as determined by monoclonal antibodies, alter during overnight storage of whole blood at 4 degrees C. In contrast, we report here that the expression of lymphocyte subpopulation markers does not alter, but that aberrant results may be obtained due to a large amount of contaminating granulocytes in the lymphocyte preparation, thus indicating that the 'apparent loss' of lymphocyte markers is a technical rather than a biological problem.     

Lymphocyte subpopulations in patients with systemic lupus erythematosus

A discrepancy between E-rosette-forming cells and total T cells identified by monoclonal antibodies (Leu-1+) was observed in patients with systemic lupus erythematosus (SLE). E-Rosette-forming cell percentages of peripheral blood mononuclear cells (MNC) were significantly lower than those observed in normal individuals, in contrast to the percentage of Leu-1+ cells in SLE patients which were not different from those of normal values. The cells which did not form E rosettes, but stained positively with the monoclonal anti-Leu-1 antibody (E-, Leu-1+) had certain functional capability evidenced by their ability to amount a proliferative response to T-cell mitogens, phytohemagglutinin and concanavalin A, but failed to provide T-helper-cell activity to support the differentiation of normal B lymphocytes into immunoglobulin-secreting cells following stimulation with pokeweed mitogen. Inhibition of E-rosette formation, but not of the staining for Leu-1, was demonstrated by incubating sera from SLE patients with normal MNC. These studies suggest that the T-cell markers, E+ and Leu-1+, do not necessarily characterize identical subpopulations of T cells. When referring to E- cells in studies with MNC from SLE patients, it should be realized that this population includes, in addition to B cells and macrophages, cells staining positively for Leu-1 antigen and reactive to T-cell mitogens.     

Prevalence of hepatitis C in patients with primary antibody deficiency

Concern over sporadic hepatitis C virus (HCV) infection from treatment with contaminated immunoglobulin in patients with immunodeficiency prompted us to screen serum from 44 patients for HCV RNA. We found no evidence of either horizontal or iatrogenic infection in our Clinic, except in two patients known to have received contaminated batches, and in one who had had regular plasma infusions. Thus, common variable immunodeficiency (CVID) patients in England do not have a high incidence of HCV infection despite exposure to various immunoglobulin products. This contrasts with the recently reported high prevalence in similar patients in Italy. Although our data reinforce the impression that both intramuscular and current licensed i.v. preparations in the UK are safe, manufacturers should be encouraged to PCR screen all batches of immunoglobulin for HCV.     

Lymphocyte subpopulations in non-neoplastic thymus from myasthenia gravis patients.

Lymphocyte populations in non-neoplastic thymuses from fifteen patients with myasthenia gravis (MG) were examined. As in normal subjects, the great majority of thymic lymphocytes of MG patients are T cells. When MG thymuses were compared to normal glands, lower percentages of lymphocytes able to form E rosettes resistant to incubation at 37 degrees C (stable E rosettes) were found in MG thymuses. A negligible B cell content was detected in eight normal and in eight MG thymuses with absent or rare lymph follicles; but there was a substantial B cell presence in the thymuses of seven MG cases with thymic hyperplasia containing many germinal centres. Normal and MG thymuses contain the same percentage of lymphocytes bearing receptors for the Fc portion of IgM (TM). Moreover, the IgM Fc receptor was found mostly on cells which did not form stable E rosettes and did not bear surface immunoglobulin. The possible significance of these findings is discussed.     

Lymphocyte subpopulations in peripheral blood of patients with non-Hodgkin lymphoma

Various lymphocyte subpopulations of peripheral blood of 65 patients with aleukaemic non-Hodgkin lymphoma were studied. The clinically active state and remission in both low and high grade malignancy groups were compared. An elevated lymphocyte count was found in the blood of aleukaemic patients, too. Lymphocytosis in low grade malignancies in the clinically active states was found to be higher than in the corresponding group of the high grade cases. The data showed that the most important factor in the increase of total blood lymphocyte count is the increase in the amount of B-lymphocytes. The TA/TT ratio was significantly reduced in all subgroups. The OKT4/OKT8 ratio was normal in all cases.     

Lymphocyte subpopulations in the skin of patients with chronic urticaria

In order to characterize the nature of the mononuclear cells in the perivascular infiltrates in the skin of 11 patients with CU, skin biopsy specimens were analyzed in situ by an avidin-biotin immunoperoxidase technique. Serial frozen sections were stained for total T cells, helper-inducer T cells, suppressor-cytotoxic T cells, B cells, monocytes/macrophages, and HLA-DR antigen. The infiltrates were found to consist mainly of T cells, whereas B cells and macrophages were rarely seen. Most of the T cells possessed the T4+ helper phenotypes, whereas smaller numbers of infiltrating cells were defined as suppressor-cytotoxic cells. Most of the helper-inducer T cells coexpressed the Ia (HLA-DR) antigen. Several potential pathogenic mechanisms could be implicated in CU based on these observations.     

Lymphocyte subpopulations in patients with hydroxocobalamin responsive megaloblastic anaemia.

Lymphocyte subpopulations and intrinsic factor and gastric parietal cell antibodies have been measured in 23 patients with megaloblastic anaemia who responded to treatment with hydroxocobalamin. The ratio of helper (OKT4) to suppressor (OKT8) lymphocytes was significantly increased in patients with intrinsic factor antibody compared with those who lacked the antibody. No such correlation was found for gastric parietal cell antibody. Alterations in the lymphocyte helper to suppressor (OKT4:OKT8) ratio may be associated with pernicious anaemia.     

Lymphocyte subpopulations in patients with viral hepatitis B and delta-infections

Changes in subpopulations of T3, T4, T8, HNK-1, Ia-positive, B-IgM and B-IgG producing lymphocytes were studied in patients with acute and chronic active virus hepatitis B and delta infection. A decrease of T4-helpers/inducers was demonstrated in all the groups under study and of T3-lymphocytes only in patients with delta-hepatitis turning into liver cirrhosis. The number of suppressors/cytotoxic T8 cells increased and T4/T8 ratio decreased in most patients. The number of HNK-1, Ia-positive, B-IgM and B-IgG lymphocytes increased in many subjects examined. Most marked quantitative changes in lymphocyte subpopulations were observed in patients with severe forms of delta-hepatitis and with liver cirrhosis. Changes in lymphocyte subpopulations in patients with viral hepatitis B and delta-infection are explained by reconstruction of the immune system in response to infection, affection of lymphocytes with viruses and emergence of antilymphocyte antibodies.     

T cells from patients successfully treated with OKT3 do not react with the T-cell receptor antibody

The mechanism of OKT3 therapy is complex and may include depletion of circulating CD3 cells, modulation of the CD3 molecule, and/or functional inactivation of T cells. Although the absolute number of circulating CD3 cells in OKT3-treated patients is used to monitor therapy, many laboratories assign CD3 numbers based on reactivity with OKT3. These CD3 numbers could be artificially low since the epitope recognized by OKT3 may already be occupied. Using a monoclonal antibody against a different CD3 epitope, we detected CD3 expression on T lymphocytes from 18/18 OKT3-treated patients. Nonetheless, OKT3 therapy in these patients was clinically successful, suggesting that monitoring patients solely for CD3 is uninformative. Since CD3 is associated with the T-cell receptor (TcR), we also evaluated alpha-TcR-1, a monoclonal antibody which detects a conformational determinant of the CD3/TcR alpha/beta complex, and found that less than 1% of the CD3 cells from OKT3-treated patients reacted. Furthermore, these cells were unresponsive to allogeneic stimulation. However, when patient cells were cultured overnight in the absence of OKT3, both alpha-TcR 1 binding and responsiveness to allogeneic stimulation became detectable. Thus, the monitoring of patients treated with OKT3 can be more informative if lymphocytes are tested for reactivity with alpha-TcR-1 and an alpha-CD3 antibody other than OKT3.     

Lymphocyte subpopulations and reactivity to mitogens in patients with scleroderma.

T lymphocyte subpopulations were studied in 40 patients with scleroderma (PSS), 26 of whom were studied simultaneously for lymphoproliferative responses to phytohaemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM). PSS patients exhibited a reduction relative to 42 age- and sex-matched controls in the absolute number and percentage of early E rosettes, late E rosettes and E rosettes formed with aminoethylisothiouronium bromide (AET) treated sheep red blood cells. There was no difference between patients and controls in the proportions of B lymphocytes. PSS patients exhibited normal lymphocyte transformation responses to PHA and ConA and an augmented response to PWM. The mitogen responses did not correlate with the absolute number or percentage of lymphocytes or T and B lymphocyte subpopulations. No correlation was observed between any immunological variable studied and the extent of skin or organ involvement, disease duration or therapy.     

Airway macrophages from patients with asthma do not proliferate

Background: Macrophages are involved in asthma, but their pulmonary turnover is unknown. We compared the ability of bronchoalveolar lavage (BAL) and bronchial macrophages to proliferate in normal subjects and patients with asthma.Methods: BAL cells from eight patients with asthma and eight normal volunteers were separated with a discontinuous Percoll gradient (Pharmacia Fine Chemicals, Uppsala, Sweden). In a first experiment, nuclei of each alveolar macrophage (AM) fraction, stained with propidium iodide, were analyzed for DNA content with a flow cytometer, and the proportions of cells in the G₀/G₁ S, and G₂ + M phases were determined. In a second experiment, expression of Ki-67-related antigen was sought on AMs by immunocytochemistry. Macrophages from 10 patients with asthma and 10 normal volunteers were studied in biopsy specimens by means of immunohistochemistry with a panmacrophage monoclonal antibody (HAM-56) and a monoclonal antibody against proliferating cell nuclear antigen.Results: The proportions of BAL AMs in the different phases of the cell cycle were similar in normal subjects and patients with asthma for all fractions, and the percentage of cells in S and G₂ ± M phases ranged from 7.3% to 11.3%. Under 1 % of BAL AMs expressed Ki-67-related antigen. None of the macrophages present in the biopsy specimens expressed proliferating cell nuclear antigen.Conclusions: This study does not indicate that an important source of airway macrophages is local proliferation.     

Meningococcal meningitis in two patients with primary antibody deficiency treated with replacement intravenous immunoglobulin

The current treatment of primary antibody deficiency (PAD) is the early recognition of the condition and replacement immunoglobulin combined with prompt treatment of infections and complications. The route of administration (intravenous or subcutaneous), dose and frequency of administration of immunoglobulin still vary between centres and countries. Most infections in patients with PAD are reduced but not entirely prevented by replacement immunoglobulin, with sinopulmonary infections accounting for the bulk of the remainder. Although there have been reports of meningitis in patients with PAD before replacement treatment, we describe the first two cases of bacterial meningitis (group B Neisseria meningitidis) on adequate immunoglobulin replacement and discuss the involvement of potential cofactors.     

Clinical implications of systematic phenotyping and exome sequencing in patients with primary antibody deficiency

PurposeThe etiology of 80% of patients with primary antibody deficiency (PAD), the second most common type of human immune system disorder after human immunodeficiency virus infection, is yet unknown.MethodsClinical/immunological phenotyping and exome sequencing of a cohort of 126 PAD patients (55.5% male, 95.2% childhood onset) born to predominantly consanguineous parents (82.5%) with unknown genetic defects were performed. The American College of Medical Genetics and Genomics criteria were used for validation of pathogenicity of the variants.ResultsThis genetic approach and subsequent immunological investigations identified potential disease-causing variants in 86 patients (68.2%); however, 27 of these patients (31.4%) carried autosomal dominant (24.4%) and X-linked (7%) gene defects. This genetic approach led to the identification of new phenotypes in 19 known genes (38 patients) and the discovery of a new genetic defect (CD70 pathogenic variants in 2 patients). Medical implications of a definite genetic diagnosis were reported in ~50% of the patients.ConclusionDue to misclassification of the conventional approach for targeted sequencing, employing next-generation sequencing as a preliminary step of molecular diagnostic approach to patients with PAD is crucial for management and treatment of the patients and their family members.     

Patients with humoral primary immunodeficiency do not develop protective anti-influenza antibody titers after vaccination with trivalent subunit influenza vaccine

Yearly influenza vaccination is recommended for patients with humoral primary immunodeficiency (hPID). However, humoral responses following vaccination can be expected to be reduced in these patients.     

Anti-D immunoglobulin treatment for thrombocytopenia associated with primary antibody deficiency

AIMS: To review our experience of anti-D immunoglobulin for immune thrombocytopenia (ITP) in patients with primary antibody deficiency. METHODS/PATIENTS: A retrospective case notes review of four Rhesus positive patients with ITP and primary antibody deficiency, treated with anti-D. Patients were refractory to steroids and high dose intravenous immunoglobulin (IVIG). Two patients were previously splenectomised. RESULTS: All patients responded to anti-D immunoglobulin. Improved platelet counts were sustained for at least three months. Side effects included a fall in haemoglobin in all cases; one patient required red blood cell transfusion. Two patients had transient neutropenia (< 1 x 10(9)/litre). CONCLUSION: Anti-D immunoglobulin may be an effective treatment for antibody deficiency associated thrombocytopenia, even after splenectomy. Anti-D immunoglobulin may have considerable clinical advantages in this group of patients, where treatments resulting in further immunosuppression are relatively contraindicated.     

Glomeruli from patients with nephrin mutations show increased number of ciliated and poorly differentiated podocytes

Background

Podocytes are postmitotic, highly specialized cells which maintain the glomerular filtration barrier (GFB). Their injury is characterized by foot processes effacement and change in protein expression leading to proteinuria and end-stage kidney disease.

T-cell subpopulations defined with monoclonal antibodies in patients with primary immunodeficiency

Peripheral blood mononuclear cells from 16 patients with common variable immunodeficiency and 4 with selective IgA deficiency were studied for the quantitative analysis of T-cells and T-cell subsets with distinct immunoregulatory properties, using a battery of monoclonal antibodies and the fluorescence-activated cell-sorter. The proportions of OKT4+ cells were decreased and OKT8+ cells were increased in patient groups when compared to normal controls analyzed simultaneously. 14/20 (70%) patients demonstrated a lower OKT4+/OKT8+ cell ratio compared to controls. Imbalance of immunoregulatory T-cells may explain one of the mechanisms of hypogammaglobulinemia in a subgroup of patients with primary immunodeficiency.     

The life situations of patients with primary antibody deficiency untreated or treated with subcutaneous gammaglobulin infusions.

The life situations of 25 patients with hypogammaglobulinaemia were studied before and after the initiation of subcutaneous replacement therapy by using medical records, data registers and questionnaires (a study and a disease-specific questionnaire, the Sickness Impact Profile and the General Health Rating Index). Before treatment, the patients perceived more dysfunctions with regard to ambulation, mobility, emotional behaviour, social interaction, sleep and rest, household management, work and recreation or pastime activities compared with a Swedish reference group (P = 0.0001). A significant increase in the perceived frequency of infections was also seen in untreated patients compared with a group of healthy individuals (P = 0.0001). After 18 months of weekly subcutaneous infusions of an intramuscular gammaglobulin preparation (100 mg/kg), the patients reported a significantly increased, health-related function and improved self-rated health. A significantly higher pre-infusion IgG level was also found.     

Lymphocyte subpopulations, serum IgE and total eosinophil counts in patients with bronchial asthma.

Absolute T and B lymphocytes, total eosinophil counts (TEC), and serum IgE levels were studied in twenty-three asthmatic subjects. Compared to a mean peripheral T-lymphocyte count of 1620+/-486/mm3 in twenty control subjects, the mean value in the asthmatic patients was depressed significantly at 1192+/-4 12 (P less than 0-005). T lymphopenia was observed in ten of twenty-three patients (43%). The serum IgE was elevated above the upper limits of 507 mug/ml in six of seventeen patients. TEC above 250/mm3 was observed in twelve of seventeen subjects. T lymphopenia and hyper-immunoglobulinaemia E were each associated with hypereosinophilia. No correlation was observed between T lymphopenia and hyperimmunoglobulinaemia E.