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Overall survival of patients with Nijmegen breakage syndrome depending on the time when they were treated due to malignancies: before and after 2000 year.

The dynamics of primary cancer incidence among patients with NBS described using the three-stage joint-point model.

Overall survival of patients with Nijmegen breakage syndrome after cancer diagnosis. Patients who underwent HSCT were marked with an asterix.

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IntroductionDeficiency of adenosine deaminase (ADA) manifests as severe combined immunodeficiency (SCID), caused by accumulation of toxic purine degradation by-products. Untreated patients develop immune and non-immune symptoms with fatal clinical course. According to ESID and EBMT recommendations enzyme replacement therapy (ERT) should be implemented as soon as possible to stabilize the patient’s general condition, normalize transaminases, treat pulmonary proteinosis, bone dysplasia, and protect from neurological damage. Hematopoietic stem cell transplantation (HSCT) from a matched related donor (MRD) is a treatment of choice. In absence of such donor, gene therapy (GT) should be considered. HSCT from a matched unrelated donor (MUD) and haploidentical hematopoietic stem cell transplantation (hHSCT) are associated with worse prognosis.Material and methodsWe retrospectively evaluated the clinical course and results of biochemical, immunological and genetic tests of 7 patients diagnosed in Poland with ADA deficiency since 2010 to 2022.ResultsAll patients demonstrated lymphopenia affecting of T, B and NK cells. Diagnosis was made on the basis of ADA activity in red blood cells and/or genetic testing. Patients manifested with various non-immunological symptoms including: lung proteinosis, skeletal dysplasia, liver dysfunction, atypical hemolytic-uremic syndrome, and psychomotor development disorders. Five patients underwent successful HSCT: 3 patients from matched unrelated donor, 2 from matched sibling donor, and 1 haploidentical from a parental donor. In 4 patients HSCT was preceded by enzyme therapy (lasting from 2 to 5 months). One patient with multiple organ failure died shortly after admission, before the diagnosis was confirmed. None of the patients had undergone gene therapy.ConclusionsIt is important to diagnose ADA SCID as early as possible, before irreversible multi-organ failure occurs. In Poland HSCT are performed according to international immunological societies recommendations, while ERT and GT are less accessible. Implementation of Newborn Screening (NBS) for SCID in Poland could enable recognition of SCID, including ADA-SCID.

DNA ligase I deficiency is an extremely rare primary immunodeficiency with only 6 patients reported in the literature. Most common manifestations include radiosensitivity, macrocytic anemia, lymphopenia with an increased percentage of gamma-delta T cells, and hypogammaglobulinemia requiring replacement therapy. Two-month-old girl with delayed development, T-B-NK+ SCID, and macrocytic anemia presented features of Omenn syndrome. Whole exome sequencing revealed two novel, heterozygous variants (c.2312 G>A, p.Arg771Gly and c.776+5G>T, p.Pro260*) in the LIG1 gene (NM_000234.1). Hematopoietic stem cell transplantation from a fully matched unrelated donor was performed at the age of 4 months using GEFA03 protocol. Mixed donor-recipient chimerism was observed, with 60-70% chimerism in the mononucleated cell compartment and over 90% in T-lymphocyte compartment, but autologous myeloid recovery. Stable CD4+ and CD8+ T-cell counts above 200/µL were achieved after 2 months, but the patient remained transfusion-dependent. Despite satisfactory immunological reconstitution, the second transplantation due to constitutional hemolytic defect has been considered. In light of possible re-transplantation, an issue of optimal conditioning protocol with sufficient myeloid engraftment is important. For the first time Omenn syndrome is described in a compound heterozygote carrying two the novel variants p.Arg771Gly and p.Pro260* in the LIG1 gene. Patients diagnosed with SCID and Omenn syndrome showing macrocytic anemia, should be screened for DNA ligase I deficiency.

Background and Purpose It is a matter of research, whether children with immunodeficiencies are able to generate an effective immune response to prevent SARS-CoV-2 reinfection. This study aimed to evaluate and compare the seroconversion rates and changes of lymphocyte subsets during COVID-19 in immunocompetent children and those with secondary immunodeficiencies. Methods In 55 children — 28 immunocompromised and 27 immunocompetent — hospitalized with confirmed SARS-CoV-2 infection, the level of IgG antibodies against the Spike protein was determined on two to three occasions. In those children from the study group whose immunosuppressive treatment did not alter during the study (n = 13) and in selected children from the control group (n = 11), flow cytometric evaluation of lymphocyte subsets was performed twice — 2 weeks and 3 months post-infection. Results Seroconversion reached 96.3% in both studied groups; however, the immunocompromised cohort achieved lower titers of detectable anti-S antibodies. There was no correlation between seroconversion or titers of antibodies and the total number of lymphocytes or their subsets. In the immunocompetent cohort, we reported a significant decrease in NK cells during the infection. In this group and the entire study population, a positive correlation was noticed between the CD4 + /CD8 + T cell ratio and the severity of COVID-19 pneumonia. Conclusions Children with secondary immunodeficiencies seroconvert in equal percentages but with a significantly lower titer of anti-S antibodies compared to their immunocompetent peers. The lower number of NK cells in the immunocompetent cohort may result from their participation in antiviral immunity, whereas reduced CD4 + /CD8 + T cell ratios among immunocompromised children may be a protective factor against a severe COVID-19.

GATA-binding protein 2 (GATA2) is a transcription factor responsible for the regulation of blood cell proliferation, differentiation, and maintenance in hematopoietic stem cells. Here, we describe successful bone marrow transplantation in a carrier of a novel GATA2 pathogenic variant who was diagnosed with immunodeficiency a few years after completion of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treatment. At the age of 4 years, the patient was diagnosed with and treated for BCP-ALL. Antileukemic therapy was complicated by pulmonary cryptococcosis. Two years after completion of the maintenance therapy, the child was consulted by an immunologist because of recurrent respiratory tract infections and an episode of sepsis. Flow cytometry revealed deep monocytopenia, lymphopenia, absence of B lymphocytes, considerably reduced NK cells, poor thymic T lymphocyte production, minor defects in T cell maturation, and absence of TCRγδ+ T cells. The presence of the likely pathogenic, heterozygous missense variant within exon 5 of GATA2 (NM_032638.5: c.1047T>G, Cys349Trp) was identified in the proband and confirmed in the father of the patient, who underwent allogeneic hematopoietic stem cell transplantation (HSCT) from a matched unrelated donor due to myelodysplastic syndrome with excess blasts at the age of 22 years. An allogeneic hematopoietic stem cell transplantation with a reduced toxicity conditioning protocol was performed using a matched sibling donor. Pre-transplant conditioning included fludarabine (5 × 30 mg/m2), treosulfan (3 × 14 g/m2), and thiotepa (10 mg/kg). Complete donor chimerism was achieved on post-transplant day 17. During the 12 months of the posttransplant observation period, she remained free from symptoms of acute or chronic graft-versus-host disease, and immunosuppressive treatment was therefore stopped. This is the second reported case of BCP-ALL in a patient with GATA2 deficiency, and the first successfully treated with a reduced-toxicity conditioning HSCT protocol. The co-occurrence of lymphoid malignancies and primary immunodeficiencies points to the role of genetic counseling and family screening for possible cancer predisposition syndromes prior to the selection of related HSCT donors.

Bronchiestasis is a common complication developing in patients with primary immunodeficiency disorders. AD GOF STAT1 defi-ciency is characterized by CMC, repeated infections, and autoimmunity. It is the most frequently diagnosed entity in a group of PIDs with CMC. Here, we present the first Polish case of a female patient with early-onset bronchiestasis accompanied by CMC and a severe course of infections who was genetically diagnosed with AD GOF1 STAT1 mutation at the age of 15.

De novo Donor Specific Antibodies (dnDSA) are associated with inferior graft outcomes. Standard immunosuppression is expected to prevent dnDSA production in low-risk patients. We have evaluated a cumulative effect of a triple immunosuppression (CNI/MMF/Pred), as well as TAC concentration and coefficient of variation on the incidence of dnDSA production. Overall, 67 transplanted patients were evaluated in retrospective (dnDSA for-cause; n = 29) and prospective (dnDSA by protocol; n = 38) groups. In the retrospective group, the eGFR value at first dnDSA detection (median interval—4.0 years post-transplant) was 41 mL/min/1.73 m2; 55% of patients presented biopsy-proven cAMR, and 41% lost the graft within next 2.4 years. Patients from the prospective group presented 97% graft survival and eGFR of 76 mL/min/1.73 m2 at 2 years follow-up, an overall incidence of 21% of dnDSA and 18% of acute (T cell) rejection. None of the patients from the prospective group developed cAMR. Median value of Vasudev score within 2 years of follow-up was not significantly higher in dsDSA negative patients, while median value of TAC C0 > 1–24 months post-transplant was 7.9 in dnDSA negative vs. 7.1 ng/mL in dnDSA positive patients (p = 0.008). Conclusion: dnDSA-negative patients presented a higher exposure to tacrolimus, while not to the combined immunosuppression.

Purpose: Nijmegen breakage syndrome (NBS) is a DNA repair disorder with a high predisposition to hematologic malignancies. Experimental Design: We describe the natural history of NBS, including cancer incidence, risk of death, and the potential effectiveness of hematopoietic stem cell transplantation (HSCT) in preventing both pathologies: malignancy and immunodeficiency. Results: Among 241 patients with NBS enrolled in the study from 11 countries, 151 (63.0%) patients were diagnosed with cancer. Incidence rates for primary and secondary cancer, tumor characteristics, and risk factors affecting overall survival (OS) were estimated. The cumulative cancer incidence was 40.21% ± 3.5% and 77.78% ± 3.4% at 10 years and 20 years of follow-up, respectively. Most of the tumors n = 95 (62.9%) were non-Hodgkin lymphomas. Overall, 20 (13.2%) secondary malignancies occurred at a median age of 18 (interquartile range, 13.7–21.5) years. The probability of 20-year overall survival (OS) for the whole cohort was 44.6% ± 4.5%. Patients who developed cancer had a shorter 20-year OS than those without malignancy (29.6% vs. 86.2%; P < 10−5). A total of 49 patients with NBS underwent HSCT, including 14 patients transplanted before malignancy. Patients with NBS with diagnosed cancer who received HSCT had higher 20-year OS than those who did not (42.7% vs. 30.3%; P = 0.038, respectively). In the group of patients who underwent preemptive transplantation, only 1 patient developed cancer, which is 6.7 times lower as compared with nontransplanted patients [incidence rate ratio 0.149 (95% confidence interval, 0.138–0.162); P < 0.0001]. Conclusions: There is a beneficial effect of HSCT on the long-term survival of patients with NBS transplanted in their first complete remission of cancer.

ObjectivesThe aim of the study was to estimate the rate of adverse reactions to live BCG Moreau vaccine, manufactured by Biomed in Poland, in severe combined immunodeficiency (SCID) patients.MaterialThe profiles of 52 SCID patients vaccinated at birth with BCG, hospitalized in Children’s Memorial Health Institute, Warsaw (CMHI), in the years 1980–2015 were compared with those of 349 BCG-vaccinated SCID patients from other countries analyzed by Beatriz E. Marciano et al. in a retrospective study (Marciano et al. J Allergy Clin Immunol. 2014;133(4):1134–1141).ResultsSignificantly less disseminated BCG infections (10 out of 52 SCID, 19%) occurred in comparison with Marciano study—119 out of 349, 34% (p = 0.0028), with no death in patients treated with SCID anti-TB drug, except one in lethal condition. In our study, disseminated BCG infection was observed only in SCID with T-B+NK- phenotype and significantly lower NK cell counts (p = 0.0161). NK cells do not influence on the frequency of local BCG reaction. A significantly higher number of hematopoietic stem cells transplantations (HSCT) were performed in CMHI study (p = 0.0001). Anti-TB treatment with at least two medicines was provided.ConclusionThe BCG Moreau vaccine produced in Poland, with well-documented genetic characteristics, seems to be safer than other BCG substrains used in other regions of the world. Importantly, NK cells seem to play a role in protecting SCID patients against disseminated BCG complications, which NK- SCID patients are more prone to. HSCT and TB therapy could be relevant due to the patients’ survival and the fact that they protect against BCG infection.

Pediatric patients with end-stage renal failure due to severe drug-resistant nephrotic syndrome are at risk of rapid recurrence after renal transplantation. Treatment options include plasmapheresis, high-dose of cyclosporine A/methylprednisolone and more recently—rituximab (anti-B CD20 monoclonal depleting antibody). We report five patients with immediate (1–2 days) post-transplant recurrence of nephrotic syndrome, treated with this kind of combined therapy including 2–4 weekly doses of 375 mg/m2 of rituximab. Only two (of five) patients have showed full long-term remission, while the partial remission was seen in two cases, and no clinical effect at all was achieved in one patient. The correlation between B CD19 cells depletion and clinical effect was present in two cases only. Severe adverse events were present in two patients, including one fatal rituximab-related acute lung injury. Conclusion: The anti-CD20 monoclonal antibody may be not effective in all pediatric cases of rapid post-transplant recurrence of nephrotic syndrome, and benefit/risk ratio must be carefully balanced on individual basis before taking the decision to use this protocol. What is Known: • nephrotic syndrome may recur immediately after renal transplantation • plasmapheresis combined with pharmacotherapy is used as rescue management • rituximab was reported as effective drug both in primary and post-transplant nephrotic syndrome What is New: • rituximab may not be effective is several cases of post-transplant nephrotic syndrome due to variety of underlying mechanisms of the disease, which may be or not be responsive to this drug • there may be no correlation between drug-induced depletion of specific B cells and clinical effect; this might suggest B-cell independent manner of rituximab action

Combined liver-kidney transplantation (CLKT) is a rare procedure in pediatric patients in which liver and kidney from 1 donor are transplanted to a recipient during a single operation. The aim of our study was to analyze indications and results of CLKT in children. Materials and Methods Between 1990 and 2017 we performed 722 liver transplantations in children; we performed 920 kidney transplantations in children since 1984. Among them, 25 received CLKT. Primary diagnosis was fibro-polycystic liver and kidney disease in 17 patients, primary hyperoxaluria type 1 in 6 patients, and atypical hemolytic uremic syndrome-related renal failure in 2 children. Age of patients at CLKT was 3 to 23 years (median 16 years) and body mass was 11 to 55 kg (median 35.5kg). All patients received whole liver graft. Kidney graft was transplanted after liver reperfusion before biliary anastomosis. Cold ischemia time was 5.5 to 13.3 hours (median 9.4 hours) for liver transplants and 7.3 to 15 hours (median 10.4 hours) for kidney transplants. In 8 patients X-match was positive. We analyzed posttransplant (Tx) course and late results in our group of pediatric recipients of combined grafts. Results Tx follow-up ranged from 1.5 to 17 years (median 4.5 years). Two patients died: 1 patient with oxalosis lost renal graft and died 2.6 years after Tx due to complications of long-term dialysis, and 1 died due to massive bleeding in early postoperative period. Twelve patients were transferred under the care of adult transplantation centers. Six patients were dialyzed after CLKT due to acute tubular necrosis, and time of kidney function recovery was 10 to 27 days in these patients. In 1 patient with aHUS, renal function did not recover. In children with oxalosis, hemodialysis was performed for 1 month after Tx as a standard, with the aim to remove accumulated oxalate. Primary immunosuppression consisted of daclizumab or basiliximab, tacrolimus, mycophenolate mofetil, and steroids. Acute rejection occurred in 4 liver and 3 kidney grafts. One patient required liver retransplantation due to hepatitis C virus recurrence and 2 patients required kidney retransplantation. Two patients required dialysis. Conclusions CLKT in children results in low rate of rejection and high rate of patient and graft survival.

The retrospective analysis of a immunological and clinical survey in 33 boys with definitive diagno sis of X-linked agammaglobulinemia, based on ESID criteria, is presented. Recurrent lower respiratory tract infections were the most common presentation with the onset at the age of 1 year 3 months, when maternal antibodies disappeared. The clinical diagnosis was usually delayed by 2 years and 6 months. The mean IgG level at diagnosis was 1.03 g/l (range 0-4.0 g/l). Mean IgA was 0.039 g/l (range 0-0.38 g/l) and IgM – 0.189 g/l (range 0.021-3.51 g/l). The total number of B cells was decreased and varied between 0-391 cells/µl, with the mean value of 26 cells/µl and 0.65%. The replacement therapy with intra venous immunoglobulin (IVIG) preparation was also delayed, and introduced at the mean age of 3 years 5 months. In some patients it was followed with subcutaneous immunoglobulin (SCIG). The overall prog nosis in XLA patients is good if diagnosis and immunoglobulin replacement therapy are done early before the onset of chronic complications. Diagnosis was established through molecular analysis.

Patients with an immunodeficiency in the course of Nijmegen breakage syndrome (NBS) that is caused by mutations in the NBN/NBS1 gene are prone to recurrent infections and malignancies, due to a defective DNA double-strand breaks repair mechanism. Four-color flow cytometry was used to analyze changes in B lymphocyte subsets reflecting the most important stages of peripheral B cell maturation. It was demonstrated that the humoral immune defect observed in NBS patients was caused by reduced numbers of B lymphocytes, but also by their aberrant maturation. Reduced relative and absolute counts of naive and memory B cells were accompanied by a significant accumulation of the natural effector B lymphocytes. The elevated proportion of IgM-only memory and reduced proportion of IgM-negative cells within the memory B cell pool suggests that there is class-switch recombination defect in this population of cells in NBS patients, resulting in inadequate production of immunoglobulins. Because of the reduced T-cell counts, the T-cell dependent antigen response is severely impaired resulting in a lower frequency of memory B-cells. The T-cell independent B-cell differentiation pathway seems less affected. The reduced IgG and IgA levels in patients with NBS are caused both by ineffective class switch, at least due to poor T cell help, and low number of memory B cells. This study illustrates that the NBN gene product nibrin plays an important role at different levels in the B-cell system. (C) 2012 International Society for Advancement of Cytometry

Background: The process of maturation of the immune system leads to generation of various lymphoid cell populations having the ability to react in specific way and expressing various markers on the cell surface. The study was set up to establish reference values for B lymphocyte subpopulations in peripheral blood of children and young adults to find the spectrum of their physiological age-related variation. Methods: Blood samples were taken from 292 children and young adults aged 0–31 years and tested for distribution of B cell subsets. Relative and absolute sizes of non-memory and memory, transitional, naive, immature marginal zone-like/IgM-only memory, class-switched memory, double negative, activated, and plasmacytoid cell populations were determined by four-color flow cytometry, based on differential expression of CD19, IgM, IgD, CD21, CD27, and CD38. Significant variation both in relative, as well as in absolute numbers of individual cell populations in tested groups was observed. Results: The reference values for age-related B cell subsets in eleven age groups, established as result of this study, may be used in diagnostics of any pathology related to B cell maturation process, as well as in attempts of correlating laboratory results with clinical symptoms of many defects affecting antibody production in pediatric population. Conclusion: Determination of B cell subpopulations carried in patients with antibody deficiencies may help to understand the nature of the disease and prevent its complications. © 2010 International Clinical Cytometry Society

Rodzinna limfohistiocytoza (FHL)/rodzinny zespol hemofagocytarny to rzadka, zagrazająca zyciu choroba, bedąca skutkiem wrodzonego upośledzenia aktywności cytotoksycznej komorek NK i limfocytow T cytotoksycznych, ktora zwykle ujawnia sie pod wplywem zakazenia wirusowego. Glownymi objawami tego dziedziczonego w sposob autosomalnie recesywny zespolu są: trwająca ponad 7 dni gorączka, powiekszenie wątroby i/lub śledziony oraz bi- lub pancytopenia we krwi obwodowej. Do typowych objawow laboratoryjnych nalezą: hipofibrynogenemia, hipertrojglicerydemia, hiperferrytynemia, hipertransaminazemia, hiperbilirubinemia oraz limfocytarna pleocytoza w plynie mozgowo-rdzeniowym. W przebiegu choroby czesto obserwowane są zaburzenia funkcji wątroby i ośrodkowego ukladu nerwowego, spowodowane hipercytokinemią i naciekami z hiperaktywowanych makrofagow i limfocytow cytotoksycznych. Hemofagocytoza – charakterystyczny objaw morfologiczny, bywa nieobecna we wcześnie pobranej biopsji szpiku lub wezla chlonnego. Jeśli kryteria diagnostyczne są spelnione, to przed wykonaniem badan genetycznych nalezy oznaczyc wewnątrzkomorkową ekspresje perforyny w komorkach NK i limfocytach T cytotoksycznych, np. za pomocą cytometrii przeplywowej. Badanie to wykonywane jest w Pracowni Zgodności Tkankowej IP-CZD w Warszawie. Wynik mozna uzyskac w ciągu dwoch godzin. Chorzy pozbawieni wewnątrzkomorkowej ekspresji perforyny wymagają przeprowadzenia diagnostyki genetycznej w poszukiwaniu mutacji w genie PRF1, odpowiadającej za okolo 40% FHL na świecie. Pelne wyleczenie mozna osiągnąc jedynie po udanym przeszczepieniu macierzystych komorek krwiotworczych.

Nijmegen Breakage Syndrome (NBS) is a rare inherited condition, characterized by microcephaly, chromosomal instability, immunodeficiency, and predisposition to malignancy. This retrospective study, characterizing the clinical and immunological status of patients with NBS at time of diagnosis, was designed to assess whether any parameters were useful in disease prognosis, and could help determine patients qualified for hematopoietic stem cell transplantation. The clinical and immunological characteristics of 149 NBS patients registered in the online database of the European Society for Immune Deficiencies were analyzed. Of the 149 NBS patients, 91 (61 %), of median age 14.3 years, remained alive at the time of analysis. These patients were clinically heterogeneous, with variable immune defects, ranging from negligible to severe dysfunction. Humoral deficiencies predisposed NBS patients to recurrent/chronic respiratory tract infections and worsened long-term clinical prognosis. Eighty malignancies, most of lymphoid origin (especially non-Hodgkin’s lymphomas), were diagnosed in 42 % of patients, with malignancy being the leading cause of death in this cohort. Survival probabilities at 5, 10, 20 and 30 years of age were 95, 85, 50 and 35 %, respectively, and were significantly lower in patients with than without malignancies. The extremely high incidence of malignancies, mostly non-Hodgkin’s lymphomas, was the main risk factor affecting survival probability in NBS patients. Because treatment of NBS is very difficult and frequently unsuccessful, the search for an alternative medical intervention such as hematopoietic stem cell transplantation is of great clinical importance.

Summary Objective Some patients suffer from recurrent or chronic rhinosinusitis (CRS) because of previously unrecognized immunodeficiency disease. The purpose of the study was to evaluate the incidence of CRS in children with different types of primary humoral deficiency. Study design Among 842 cases of primary immunodeficiency diseases diagnosed in the Department of Immunology of the Children's Memorial Institute in Warsaw from 1980 up to February 2006 antibody deficiencies occurred most frequently—in 50.4%. In this group the retrospective study of patients’ history suggesting CRS prior to intravenous gamma-globulin substitution therapy was performed. Results CRS was observed most frequently in the group of patients with agammaglobulinemia less often in patients with dysgammaglobulinemia, and other antibody deficiencies. Conclusions The occurrence of frequent bacterial infections, complicating common colds, should alert the physician to the possibility of immunodeficiency. Immunological testing should be an integral part of evaluation of patients with refractory CRS.

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.

Background: Defects in early B lymphocyte maturation in bone marrow (BM) compose a characteristic feature of many primary immune deficiencies associated with agammaglobulinemia. To date, only limited data on the composition of the precursor B-cell compartment in BM is available. The aim of this study was to define normal age-related ranges of total B-cell content and distribution of precursor B—cell stages in BM for the future use in clinical diagnostics. Methods: Four color flow cytometry was used to analyze the composition of the B-cell compartment in specimens from 59 hematologically healthy children, aged 14 days to 16 years, assigned to six age groups: neonates less than 1 month old, infants >1‐12 months old, children >1‐2 years old, >2‐5 years old, >5‐10 years old, and older than 10 years. Results: Analysis of the composition of the B-cell compartment revealed significant age-related variation in the distribution of individual B-cell maturation stages, most seriously affecting children during first 2 years of life, with the shift from domination of the earliest stages, to gradually increasing content of mature B-cells. Significantly higher proportions of pro-B lymphocytes were observed in neonates than in any other age group. Conclusion: Physiological age-related variation in the precursor B-cell compartment composition affects most seriously very young children below the age of 2 years. Proper interpretation of immunophenotyping results performed in cases of suspected early B-cell differentiation defect requires application of adequate reference data. V C 2013 International Clinical Cytometry Society Key terms: precursor B-cell compartment; bone marrow; flow cytometry

BACKGROUND Despite prospective crossmatching and modern immunosuppression, early acute rejection is still present in cadaveric renal transplantation. The purpose of this study was to evaluate the incidence of preformed anti-donor antibodies, detected by 2 solid-phase techniques, and to analyze their impact on early renal allograft outcome. MATERIAL/METHODS Flow crossmatch detecting the presence of anti-donor IgG and IgM antibodies was performed in pre-transplant sera of 279 patients with negative cytotoxic crossmatch. Screening for IgG antibodies detected by bead-based multiplex technique was performed in sera of 69 patients from the FCXM group. The incidence of early biopsy-proven rejection and graft failure within 3 months after transplantation was analyzed. RESULTS Anti-donor IgG antibodies were detected in 33 patients (11.8%) by flow crossmatch and in 10 patients by multiplex (14.5%). IgM antibodies were detected in 23 patients (8.2%). All multiplex-positive sera were also positive for IgG by flow crossmatch, but in 18 cases no antibodies were found by multiplex technique. Biopsy-proven acute rejection within 3 months after transplantation was observed in 16 patients, and 5 allografts were lost due to immunological reasons. Presence of IgG antibodies was found to have no effect on early outcome, while the presence of IgM antibodies was associated with significantly higher rejection rate and immune-related graft failure. CONCLUSIONS Anti-donor IgG antibodies detected by bead-based and cell-based technique have no impact on biopsy-proven rejection rate or graft failure. Anti-donor IgM detected by flow crossmatch have significant impact on early transplantation outcome.

Early recurrence of nephrotic syndrome after renal transplantation is a common serious adverse event in children with severe primary FSGS, affecting long-term outcome. There is no consensus in terms of uniform management in these cases. We describe the long-term effect of four unadjusted doses of 375 mg/m(2) i.v. rituximab, given to a five and a half-yr-old, nephrectomized child with immediate recurrence of nephrotic syndrome post-transplantation and dependency from repeated PF. Rituximab was introduced at three months post-transplantation after performing 18 sessions of PF and development of established dependency of the disease from plasma exchange. Complete remission of proteinuria was achieved with four doses, and it was maintained during next eight months of follow-up. Complete B CD(19) cell depletion was observed during four months after final dose, followed by severe depletion after eight months. No side effects of therapy were noted. Patient was free from PF, which was stopped while introducing rituximab, remaining non-proteinuric on triple immunosuppression (CsA, MMF, Pred).

Grenda R, Jarmuzek W, Piątosa B, Rubik J. Long-term effect of rituximab in maintaining remission of recurrent and plasmapheresis-dependent nephrotic syndrome post-renal transplantation – Case report. Pediatr Transplantation 2011: 15: E121–E125. © 2010 John Wiley & Sons A/S. Abstract: Early recurrence of nephrotic syndrome after renal transplantation is a common serious adverse event in children with severe primary FSGS, affecting long-term outcome. There is no consensus in terms of uniform management in these cases. We describe the long-term effect of four unadjusted doses of 375 mg/m2 i.v. rituximab, given to a five and a half-yr-old, nephrectomized child with immediate recurrence of nephrotic syndrome post-transplantation and dependency from repeated PF. Rituximab was introduced at three months post-transplantation after performing 18 sessions of PF and development of established dependency of the disease from plasma exchange. Complete remission of proteinuria was achieved with four doses, and it was maintained during next eight months of follow-up. Complete B CD19 cell depletion was observed during four months after final dose, followed by severe depletion after eight months. No side effects of therapy were noted. Patient was free from PF, which was stopped while introducing rituximab, remaining non-proteinuric on triple immunosuppression (CsA, MMF, Pred).

Common variable immune deficiency (CVID) is a heterogeneous disease associated with ineffective production of antibodies. It is usually diagnosed in adulthood, but a variable proportion of children develop CVID. Early identification of patients with potentially worse prognosis may help to avoid serious complications. The goal of this study was to associate the clinical phenotype of patients with early onset CVID with peripheral B-cell maturation profile. Four color flow cytometry was used to define distribution of peripheral B-cell subsets in 49 children with early-onset CVID. All clinical data were extracted from medical records. A proportion of patients demonstrated diminishing with time total B-lymphocytes pool, beyond physiological age-related changes. Irrespective from duration of the follow-up period the B-cell maturation profile in individual patients remained unchanged. We identified six different aberrant peripheral B cell maturation profiles associated with different clinical characteristics. Patients with an early B-cell maturation block earlier required replacement therapy and were at significantly greater risk of enteropathy, granuloma formation, cytopenia, and lymphoproliferation. B-cell maturation inhibited at the natural effector stage was associated with higher risk of autoimmune manifestations other than autoimmune cytopenia. Prevalence of male patients was observed among patients with B-cell maturation inhibited at naive B-cell stage. In conclusion, the diagnostic process in patients with suspected early-onset CVID shall include routine analysis of peripheral B-cell maturation to provide surrogate markers identifying patients at greater risk of developing certain complications.

BACKGROUND The aim of our study was to retrospectively assess any correlation between graft fibrosis and selected immunological factors in pediatric liver transplant recipients. MATERIAL AND METHODS The study was performed on 33 patients after living related donor transplantation, divided into 2 groups depending on history of acute rejection episodes after transplantation. We assessed liver biopsies for presence of fibrosis, signs of antibody-mediated rejection, inflammatory infiltrations, and changes in bile ducts. We correlated these findings with assessment of anti-HLA antibodies. RESULTS Among 14 patients with ACR, a history fibrosis was found in 8 patients (57%). In 19 patients without a history of ACR, fibrosis was found in 9 patients (47%). Anti-HLA antibodies were found in 47% of patients with fibrosis and in only 18.75% of patients without fibrosis. Among 3 patients with signs of antibody-mediated rejection, all had fibrosis in the graft 2 years after transplantation. We did not find any patient with chronic rejection or ductopenia. CONCLUSIONS We suggest that there is a correlation between ACR and development of graft fibrosis present in liver grafts from recipients with normal liver biochemistry. Anti-HLA antibodies class II seems to be most important in development of fibrosis.

Background Antibody-mediated rejection (AMR), associated with the presence of C4d deposits, is well-defined in kidney transplantation but much less documented in liver transplantation (LTx). The aim of our study was to retrospectively analyze a group of pediatric liver transplant recipients who experienced episodes of acute rejection in the past, for the signs of AMR and its impact on liver histology. Material/methods Our study population consisted of 18 patients after living related donor liver transplantation with a history of acute cellular rejection (1-5/patient). In all of them, actual liver function was good at almost 2-year median follow-up after transplantation. We reassessed all liver biopsies taken from these children between 5 days to 5.7 years after transplantation for signs of acute cellular rejection and antibody-mediated rejection. In all patients, anti-HLA antibodies were also assessed at least 2 years after transplantation (2.18-12.27 years, median 6.795 years). Results There were 27 episodes of acute rejection proved by liver biopsy. Signs of AMR were found in 6 of 18 patients (33.3%). In 5 of these patients, donor-specific (DSA) and non-specific anti-HLA antibodies were also identified. In the group of 12 patients with acute rejection without histochemical signs of AMR, anti-HLA antibodies were found in sera of only 5 of 12 patients after transplantation. Conclusions Our study shows some correlation between C4d-positive reaction in liver biopsies with acute cellular rejection and presence of anti-HLA antibodies, particularly against HLA class II. We did not find any difference in the late graft function, which could be correlated with the presence of AMR. Further studies on larger groups of patients are necessary.