The Use of Induced Pluripotent Stem Cells to Study the Effects of Adenosine Deaminase Deficiency on Human Neutrophil Development.

Inherited defects that abrogate the function of the adenosine deaminase (ADA) enzyme and consequently lead to the accumulation of toxic purine metabolites cause profound lymphopenia and severe combined immune deficiency. Additionally, neutropenia and impaired neutrophil function have been reported among ADA-deficient patients. However, due to the rarity of the disorder, the neutrophil developmental abnormalities and the mechanisms contributing to them have not been characterized. Induced pluripotent stem cells (iPSC) generated from two unrelated ADA-deficient patients and from healthy controls were differentiated through embryoid bodies into neutrophils. ADA deficiency led to a significant reduction in the number of all early multipotent hematopoietic progenitors. At later stages of differentiation, ADA deficiency impeded the formation of granulocyte colonies in methylcellulose cultures, leading to a significant decrease in the number of neutrophils generated from ADA-deficient iPSCs. The viability and apoptosis of ADA-deficient neutrophils isolated from methylcellulose cultures were unaffected, suggesting that the abnormal purine homeostasis in this condition interferes with differentiation or proliferation. Additionally, there was a significant increase in the percentage of hyperlobular ADA-deficient neutrophils, and these neutrophils demonstrated significantly reduced ability to phagocytize fluorescent microspheres. Supplementing iPSCs and methylcellulose cultures with exogenous ADA, which can correct adenosine metabolism, reversed all abnormalities, cementing the critical role of ADA in neutrophil development. Moreover, chemical inhibition of the ribonucleotide reductase (RNR) enzyme, using hydroxyurea or a combination of nicotinamide and trichostatin A in iPSCs from healthy controls, led to abnormal neutrophil differentiation similar to that observed in ADA deficiency, implicating RNR inhibition as a potential mechanism for the neutrophil abnormalities. In conclusion, the findings presented here demonstrate the important role of ADA in the development and function of neutrophils while clarifying the mechanisms responsible for the neutrophil abnormalities in ADA-deficient patients.

Secondary hypogammaglobulinemia in Waldmann's disease treated with subcutaneous immunoglobulins.

Primary intestinal lymphangiectasia (PIL) is rare disorder characterized by congenital malformation or obstruction of intestinal lymphatic drainage; it is responsible for protein losing enteropathy leading to lymphopenia, hypoalbuminemia and hypogammaglobulinemia. A low-fat diet associated with medium-chain triglyceride supplementation is the cornerstone of PIL management. The administration of intravenous immunoglobulins does not always lead to satisfactory plasma levels and therefore the replacement therapy with immunoglobulins is controversial. We describe here the case of a patient with PIL and severe hypogammaglobulinemia treated with immunoglobulins. The striking aspect of this case is the clinical and serological benefit obtained with the subcutaneous compared to the intravenous immunoglobulins administration.

A novel treatment in X-linked agammaglobulinaemia - hyperbaric oxygen therapy in refractory chronic wounds.

Chronic wounds are a rare complication of X-linked agammaglobulinaemia (XLA). Fastidious organisms such as helicobacter bills have been reported in XLA with chronic wounds but sterile chronic wounds also occur. Hyperbaric Oxygen Therapy has been used in chronic wounds but has not previously been reported in primary antibody deficiencies. We present a case of a chronic wound in a patient with XLA refractory to antimicrobial therapy that made a remarkable recovery following Hyperbaric Oxygen Therapy.

The efficacy of Pelargonium sidoides in the treatment of upper respiratory tract infections in children with transient hypogammaglobulinemia of infancy.

Transient hypogammaglobulinemia of infancy (THI), defined as prolongation of physiological hypogammaglobulinemia normally seen between the initial 3rd and 6th months of life, is one of the most common immune deficiencies of childhood. Recurrent upper respiratory tract infections (URTI) are rather common in this group of patients, and generally, antibiotic treatment is the usual choice, although viruses involved in most cases. Pelargonium sidoides extract a herbal drug with known immunmodulator, antiviral and antibacterial effects. In this randomized, placebo controlled, prospective, monocentric pilot study, 14 of 28 patients with a diagnosed THI, were given Pelargonium sidoides, while 14 were given placebo during the period of URTI. Before and after the treatment period of one week, complete blood count, prothrombin time, activated prothromboplastin time, serum alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transpeptidase, total and direct bilirubin levels were measured. Mothers were asked to fill in a questionnaire for the recovery of the clinical symptoms during the treatment. The results were evaluated and compared in both group to assess the effect of Pelargonium sidoides. As a conclusion, the Pelargonium sidoides group showed increased appetite. The Pelargonium sidoides were found to beneficial for the nasal congestion, recovery of daily and nocturnal cough but not found be significant. Further studies with large number of participants are necessary to highlight the effect of Pelargonium sidoides in children with transient hypogammaglobulinemia of infancy.

Consequences of two naturally occurring missense mutations in the structure and function of Bruton agammaglobulinemia tyrosine kinase.

Bruton agammaglobulinemia tyrosine kinase (BTK) is a key protein in the B-cell receptor (BCR) signaling pathway and plays an essential role in the differentiation of B lymphocytes. X-linked agammaglobulinemia (XLA) is a primary humoral immunodeficiency caused by mutations in the gene encoding BTK. Previously, we identified two novel variations, L111P and E605G, in BTK; these are localized within the pleckstrin homology and Src homology 1 domains, respectively. In the present study, we evaluated the potential effects of these variations on the structural conformation and the function of BTK. Using in silico methods, we found that the L111P and E650G variations are not located directly in protein-protein interfaces but close to them. They distorted the native structural conformation of the BTK protein, affecting not only its geometry and stability but also its ability for protein recognition and in consequence its functionality. To confirm the results of the in silico assays, WT BTK, L111P, and E650G variants were expressed in the BTK-deficient DT40 cell line. The mutant proteins exhibited an absence of catalytic activity, aberrant redistribution after BCR-crosslinking, and deficient intracellular calcium mobilization. This work demonstrates that L111 and E605 residues are fundamental for the activation and function of BTK.

Retrospective analysis of weekly intravenous immunoglobulin prophylaxis versus intravenous immunoglobulin by IgG level monitoring in hematopoietic stem cell transplant recipients.

Patients undergoing allogeneic hematopoietic stem cell transplant (allo HCT) have a higher incidence of infections partly due to secondary hypogammaglobulinemia. We evaluated the role of IVIG in allo HCT patients who received prophylactic IVIG 200 mg/kg once weekly regardless of IgG level (Group 1, n = 115) compared with patients who received IVIG based on IgG level <400 mg/dL (Group 2, n = 114). Primary endpoints were the utilization of IVIG, incidence of veno-occlusive disease (VOD), graft-versus-host disease (GVHD), and documented infections within the first 100 days after allo HCT. Patients in both groups were similar except for a higher number of matched unrelated donor (MUD) transplants in Group 2 (62 vs. 41, P = 0.01). There were no significant differences in the incidence all grades of GVHD (55 vs. 50), VOD (2 vs. 0) or infections in the two groups except for a higher incidence of para-influenza infections in group 1 (9 vs. 0, P = 0.003) coinciding with the flu season. We recommend monthly monitoring of IgG level and replacement only if IgG level is <400 mg/dL.

Bruton's tyrosine kinase is essential for human B cell tolerance.

Most polyreactive and antinuclear antibodies are removed from the human antibody repertoire during B cell development. To elucidate how B cell receptor (BCR) signaling may regulate human B cell tolerance, we tested the specificity of recombinant antibodies from single peripheral B cells isolated from patients suffering from X-linked agammaglobulinemia (XLA). These patients carry mutations in the Bruton's tyrosine kinase (BTK) gene that encode an essential BCR signaling component. We find that in the absence of Btk, peripheral B cells show a distinct antibody repertoire consistent with extensive secondary V(D)J recombination. Nevertheless, XLA B cells are enriched in autoreactive clones. Our results demonstrate that Btk is essential in regulating thresholds for human B cell tolerance.

IL-9-induced expansion of B-1b cells restores numbers but not function of B-1 lymphocytes in xid mice.

Mice expressing the X-linked immunodeficiency (xid) mutation lack functional Bruton's tyrosine kinase and were shown to be specifically deficient in peritoneal B-1 lymphocytes. We have previously shown that IL-9, a cytokine produced by TH2 lymphocytes, promotes B-1 cell expansion in vivo. To determine whether IL-9 overexpression might compensate the xid mutation for B-1 lymphocyte development, we crossed xid mice with IL-9-transgenic mice. In this model, IL-9 restored normal numbers of mature peritoneal B-1 cells that all belonged to the CD5(-) B-1b subset. Despite this normal B-1 lymphocyte number, IL-9 failed to restore classical functions of B-1 cells, namely, the production of natural IgM Abs, the T15 Id Ab response to phosphorylcholine immunization, and the antipolysaccharide humoral response against Streptococcus pneumoniae. By using bromelain-treated RBC, we showed that the antigenic repertoire of these IL-9-induced B-1b lymphocytes was different from the repertoire of classical CD5(+) B-1a cells, indicating that the lack of B-1 function by B-1b cells is associated with distinct Ag specificities. Taken together, our data show that B-1b cell development can restore the peritoneal B-1 population in xid mice but that these B-1b cells are functionally distinct from CD5(+) B-1a lymphocytes.

Studies on the mechanisms of allergen-induced activation of the classical and lectin pathways of complement.

Allergen extracts are efficient activators of the complement system trough the classical pathway. Involvement of the lectin pathway was not previously studied. To further examine the mechanism of complement activation by allergens, in vitro experiments, which covered early steps both of classical and lectin pathways, were performed. Two types of allergens used in these studies: parietaria (PA) and house dust (HD) mite extracts. These allergen extracts bound to the globular head of C1q and interacted with purified mannan-binding lectin (MBL) as measured by solid-phase ELISA. None of the allergen extracts was able to activate human C1 in vitro, as measured by the determination of the split products of C1s in a reconstituted precursor C1 preparation. Neither the HD nor the PA extracts induced C4d generation above background in the serum of three subjects with hypogammaglobulinaemia but normal complement haemolytic activity. After reconstitution to normal level with purified human IgG, allergen extracts induced C4d formation above control at a level comparable to that measured in normal serum incubated with the same amounts of the extracts. HD-induced C4d generation was about the same comparable in MBL-depleted serum and in normal sera. In contrast PA induced no C4d formation in the MBL-depleted serum, whereas reconstitution with purified MBL restored C4d generation. These in vitro findings indicate that although the allergen extracts can bind purified C1q and MBL, they require IgG for efficient complement activation. Depending on the allergens, this activation may be initiated through C1, MBL, or both.

Bromelain is an accelerator of phagocytosis, respiratory burst and Killing of Candida albicans by human granulocytes and monocytes.

OBJECTIVE: The aim of this study was to examine the influence of immuno modulating agents like bromelain and trypsin (e.g. Wobenzym on granulocyte and monocyte functions in healthy volunteers and patients with disorders of the humoral immuno system X-linked agammaglobulinaemia (XLA) and common variable immuno deficiency (CVID) and to find out whether the unspecific immunity could be improved by these enzymes. METHODS: In a whole-blood assay kinetics of phagocytosis, respiratory burst and killing (PBK) were measured in blood samples incubated with and without bromelain and trypsin (B/T) using Candida albicans as target organism. The time-reaction curves were analysed determining their gradient (T1) and their onset (T2) as well as the half effect time (HET). RESULTS: Phagocytes from patients with XLA showed a significantly accelerated basal phagocytosis (reduction of HET by 24% p < 0.001) compared to healthy controls. After incubation with B/T (10 microg/ml each) speed of phagocytosis was nearly doubled (phagocyte activity p < 0.0001, Candida uptake p < 0.003), T2 of respiratory burst was reduced by 65 % (p < 0.0001) and killing was accelerated by 27% (p < 0.046). However, the maximal activities of all kinetics were not altered. Incubation of phagocytes from healthy controls with B/T accelerated phagocytosis to a level comparable to that of untreated phagocytes from patients with XLA and also accelerated reactive oxygen species (ROS) production (reduction of HET by 28%, p < 0.012). In contrast to phagocytes from patients with XLA, phagocytes of patients with CVID showed a similar stimulation by B/T like healthy controls. Further experiments with the single substances showed that bromelain was the active compound. CONCLUSION: Our data suggest, that bromelain possesses immuno stimulatory properties. Phagocytes of XLA patients appear to be particularly susceptible to this stimulation.

Passive transfer of colostral immunoglobulin G in neonatal llamas and alpacas.

OBJECTIVE: To evaluate precolostral hypogammaglobulinemia in neonatal llamas and alpacas, to determine when postcolostral peak serum IgG concentrations develop, to determine whether differences in postcolostral serum IgG concentrations between llamas and alpacas exist, and to determine postcolostral half-life of serum IgG in llamas and alpacas. DESIGN: Prospective observational study. ANIMALS: 29 llama and 10 alpaca crias. PROCEDURE: Blood samples were collected prior to suckling and on days 1, 2, and 3 after parturition and analyzed for serum IgG concentration by use of a commercial radial immunodiffusion assay. Additional samples were collected on days 8, 13, and 18 from 8 crias to determine mean half-life of IgG. RESULTS: Llamas and alpacas are born severely hypogammaglobulinemic. Mean serum IgG concentrations for day-1, -2, and -3 samples for llamas were 1,578 mg/dl, 1,579 mg/dl, and 1,401 mg/dl, respectively, and for alpacas were 2,024 mg/dl, 1,806 mg/dl, and 1,669 mg/dl, respectively. Peak serum immunoglobulin concentration developed between days 1 and 2. Mean half-life of IgG for all crias was 15.7 days. CONCLUSIONS AND CLINICAL RELEVANCE: Although increased mortality has been linked to failure of passive transfer, it is clearly possible to raise crias that have low serum immunoglobulin concentrations. Llamas and alpacas do not differ significantly with respect to immunoglobulin absorption or IgG concentration in neonates. The optimal sampling time for passive transfer status is between 1 and 2 days.

An unusual case of refractory Campylobacter jejuni infection in a patient with X-linked agammaglobulinemia: successful combined therapy with maternal plasma and ciprofloxacin.

An unusual hippurate-negative strain of Campylobacter jejuni caused a chronic refractory infection in a patient with X-linked agammaglobulinemia; this infection persisted for > 2 years despite therapy with various antibiotics and immunoglobulins (Igs). To characterize the defense status of this patient, several in vitro studies, including those with T cells and polymorphonuclear leukocytes (PMNLs), were performed. T cell responses specific for C. jejuni were only weak in this patient. Chemiluminescence and bacterial killing studies with PMNLs revealed that the bactericidal activity of PMNLs against Campylobacter was enhanced more vigorously by maternal serum than by commercial Ig preparations. On the basis of these results, combined treatment with ciprofloxacin and maternal plasma was initiated, and the C. jejuni infection was rapidly cured. This case report shows that in vitro immunologic assays may be useful for characterizing immune functions of patients with chronic or refractory C. jejuni infections, thus leading to individual treatment strategies.

Extracts of wheat gluten activate complement via the alternative pathway.

We studied the ability of wheat gluten and its subfractions to activate complement directly. A sensitive sandwich ELISA employing a monoclonal antibody (MoAb) to a C9 neoepitope exposed in the terminal complement complex (TCC), a functional haemolytic assay for C5b6 generation, and Laurell's electrophoretic method of estimating C3 conversion to C3bi were used. On a weight-for-weight basis, enzyme solubilized Frazer's fraction three of gluten (FIII) produced approximately 75% of the complement activation seen with the potent activator zymosan. By contrast, activation with whole insoluble undigested gluten was very weak and similar to that seen with ovalbumin or beta-lactoglobulin. The results were the same using normal human serum or sera from patients with coeliac disease, dermatitis herpetiformis, or hypogammaglobulinaemia as the complement source. Activation by both zymosan and FIII was blocked in 0.01 M EDTA, but not in 0.01 M EGTA with 0.0025 M magnesium chloride. Zymosan and FIII activated complement in a serum from a patient with an intact alternative pathway but classical pathway haemolytic activity (CH50) of zero. Preferential heat inactivation of the alternative pathway inhibited both zymosan- and FIII-induced activation. Our results confirm that FIII is a strong activator of the alternative pathway. We discuss how gluten enteropathy might be initiated by complement.

[Reduction of pain perception of chronically ill children by intermittent infusion therapy]. / Minderung von Schmerzerlebnissen chronisch kranker Kinder bei intermittierender Infusionstherapie.

Chronically ill children who require life-long i.v. infusions in regular intervals (i.e. Agammaglobulinemia, HIV-infection) can be traumatized by such painful procedures. Some children gradually accept such infusions, whereas other children become sensitized and react with increasing fear and resistance. We report on our experience with a combined method of local anaesthesia and active role-plays enacting the i.v. infusion on a puppet. In more than 230 applications (approximately 11 per child) in our outpatient clinic, 19/20 children reported significantly less pain sensations after the 2nd or 3rd application. The children learned to handle actively the threatening situation. The method of local anaesthesia with Lidocaine/Prilocaine and role playing is suitable to relieve pain and reduces distress of such invasive events as regular i.v. infusions are.

Protection of agammaglobulinemic piglets from porcine rotavirus infection by antibody against simian rotavirus SA-11.

Rotavirus, a double-stranded RNA virus, has been implicated as a diarrhea-provoking agent in a variety of animal species. Several previous reports have shown that immunization with a single serotype may result in increased in vitro neutralization titers against serotypes not represented in the immunogen. This study was undertaken to determine whether antibody from cows immunized against simian rotavirus strain SA-11 (which is alien to pigs) could protect neonatal piglets from infection with a North Carolina isolate of porcine rotavirus. Accordingly, cows were immunized with SA-11 and an immunoglobulin G (IgG)-rich fraction was isolated from their colostrum. An IgG-rich fraction was similarly isolated from colostrum of nonimmunized cows. At equal concentrations, IgG from SA-11-immunized cows had two- to fourfold higher neutralization titers to seven of eight test strains of rotavirus, including SA-11 (serotype 3); human rotavirus serotypes 1, 3, and 4; North Carolina porcine rotavirus (serotype undetermined); Ohio State porcine rotavirus (serotype 5); and bovine rotavirus (serotype 6). The IgG-rich fractions were fed as dietary supplements to agammaglobulinemic piglets infected with the North Carolina porcine rotavirus. IgG from the SA-11-immunized cows was about eightfold more effective in protecting piglets than was IgG from nonimmunized cows.

[Agamma-, hypogamma- and hypergammaglobulinemia. The significance of the 'relative immune status' for the young calf]. / Agamma-, hypogamma- en hypergammaglobulinaemie. De betekenis van de 'relatieve immuunstatus' voor het jonge kalf.

Calves affected with hypergammaglobulinaemia are more likely to survive than are those with agamma- or hypogammaglobulinaemia. Nevertheless, there are farms on which satisfactory results are obtained in rearing calves with low Ig levels. Management conditions, including the presence of specific or non-specific, bacterial or viral infections, are important factors in these cases. Besides humoral immunity, local protection against neonatal diarrhoea afforded by colostrum, is of even greater importance. As the concentrations of Ig in young calves decrease, production of immunity in these calves will start earlier in life, which may even be within a few days.

Activation of the classical complement pathway by a polysaccharide from sugar cane.

The effects of an immunostimulating polysaccharide, Bo, from sugar cane, on the complement system have been investigated. Bo, a glucan of about 10,000 mol wt, was found to activate complement in whole human and guinea pig serum in vitro by the classical pathway. Complement consumption was also demonstrated in guinea pigs upon intravenous injection. Specifically, C1 is activated, and C4 and C2, as well as C3, are consumed. The activation is prevented when Ca++ ions are chelated by ethyleneglycoltetraacetic acid, and when C1q is lacking. Hence, it does not rest on direct activation of C1s. Supplementation of C1q-deficient human serum with purified C1q restores the ability to be activated by Bo. The alternative pathway of complement is little if at all affected by the polysaccharide. The activation of C1 seems to be mediated by immune complex formation between Bo and naturally occurring immunoglobulins. Complement in sera from two severely hypogammaglobulinemic patients was not activated by Bo, but was made reactive by addition of purified human immunoglobulin G.

The relationship between serum immunoglobulin values and incidence of respiratory disease and enteritis in calves.

Serum immunoglobulin levels were tested in 193 neonatal calves by use of the glutaraldehyde coagulation test. Hypo- and agammaglobulinaemia was found in 26.4% of the calves. A close relationship was found between immunoglobulin levels and the incidence of enteritis and mortality, while the number of calves suffering from both enteritis and respiratory disease was higher in immunoglobulin-deficient calves. The application of specific preventive measures to immunoglobulin-deficient calves is discussed.

Red blood cell associated IgG in normal and pathologic states.

We studied the anti-IgG-induced agglutination of both normal and abnormal red blood cells (RBC) using a sensitive, automated antiglobulin test. Normal RBC agglutinated strongly with anti-IgG antibody, indicating that IgG was present on the erythrocyte membrane. Young RBC, recovered by centrifugation from a normal RBC population, agglutinated with anti-IgG less than the old cells, suggesting that immunoglobulin G accumulated gradually on the RBC membrane in vivo. The degree of anti-IgG-induced RBC agglutination correlated negatively with the reticulocyte count and positively with the concentration of plasma IgG. RBC from patients with hypogammaglobulinemia appeared to have a low subnormal quantity of membrane-bound IgG, whereas the reverse was the case in hypergammaglobulinemia. During hemolytic episodes, RBC of patients with hereditary spherocytosis agglutinated poorly with anti-IgG, apparently due to predominance of young RBC. RBC of patients with nonspherocytic. Coombs-negative, nonimmune hemolytic anemia usually also agglutinated poorly with anti-IgG. However, in some cases of active hemolytic anemia, decreased agglutination with anti-IgG was not observed, suggesting that these young RBC had increased amounts of membrane-bound IgG.

Immunodeficiency, malabsorption and secretory diarrhea. A new syndrome.

Described here is a patient with severe watery diarrhea associated with common variable immunodeficiency. Malabsorption for fat, bile acids, vitamin B12 and xylose was demonstrated, but the patient failed to respond to all the usual therapeutic maneuvers. The diarrhea responded only to high dose steroid therapy. Intestinal perfusion studies showed a hitherto undescribed, presumably acquired, glucose-stimulated water, sodium and chloride secretion in the jejunum and ileum, whereas normal fluid and electrolyte transport occurred from bicarbonate and mannitol solutions. Glucose absorption itself was normal and no hormonal, morphologic or biochemical defect was demonstrated to account for the phenomenon. The patient was also interesting when compared with other patients with common variable immunodeficiency in having normal plasma cells in the intestinal mucosa and an extensive family involvement.