Prior exposure to alkylating agents negatively impacts testicular organoid formation in cells obtained from childhood cancer patients.

STUDY QUESTION: Can human pre- and peri-pubertal testicular cells obtained from childhood cancer patients, previously treated with chemotherapy, form testicular organoids (TOs)? SUMMARY ANSWER: Organoid formation from testicular tissue collected from childhood cancer patients positively correlates with SRY-Box transcription factor 9 (SOX9) expression in Sertoli cells, which in turn negatively correlates with previous exposure to alkylating chemotherapy. WHAT IS KNOWN ALREADY: Pre- and peri-pubertal boys exposed to highly gonadotoxic therapies can only safeguard their fertility potential through testicular tissue cryopreservation. Today, there is no established clinical tool to restore fertility using these testicular samples. Organoids hold promise in providing fundamental early insights in creating such platforms. However, the generation of TOs that closely resemble the innate testis, to enable a thorough monitoring of the necessary steps for germ cell differentiation and somatic functionalities, remains a challenge. STUDY DESIGN SIZE DURATION: We used a Matrigel-based three-layer gradient culture system to generate human TOs and to reveal whether chemotherapy exposure affects TO formation capacity and the functionality of pre- and peri-pubertal testicular somatic cells. Testicular cells of 11 boys (aged 7.7 ± 4.1 (mean ± SD) years) were assessed for TO formation in relation to previous chemotherapy exposure and SOX9 expression in histological sections of paraffin-embedded testicular tissue samples collected on the day of biopsy and compared with testicular tissue samples obtained from 28 consecutive patients (aged 6.9 ± 3.8 (mean ± SD) years). All 39 patients were part of the fertility preservation project NORDFERTIL; an additional 10 samples (from boys aged 5.5 ± 3.5 (mean ± SD) years, without an underlying pathology) in an internal biobank collection were used as controls. PARTICIPANTS/MATERIALS SETTING METHODS: We obtained 49 testicular tissue samples from boys aged 0.8-13.4 years. Fresh samples (n = 11) were dissociated into single-cell suspensions and applied to a three-layer gradient culture system for organoid formation. Histological sections of another 28 samples obtained as part of the fertility preservation project NORDFERTIL, and 10 samples from a sample collection of a pathology biobank were used to evaluate the effects of prior exposure to alkylating agents on testicular samples. Testicular organoid formation was defined based on morphological features, such as compartmentalized structures showing cord formation, and protein expression of testicular cell-specific markers for germ and somatic cells was evaluated via immunohistochemical staining. Hormone secretion was analysed by specific enzyme-linked immunosorbent assays for testosterone and anti-Müllerian hormone (AMH) production. MAIN RESULTS AND THE ROLE OF CHANCE: Our results revealed that 4 out of 11 prepubertal testicular samples formed TOs that showed compartmentalized cord-like structures surrounded by interstitial-like areas and increasing levels of both testosterone as well as AMH over a 7-day culture period. We observed that SOX9 expression was correlated positively with TO formation. Moreover, exposure to alkylating agents before biopsy was inversely correlated with SOX9 expression (P = 0.006). LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: Due to the limited amount of material available, only 11 out of the 39 pre- and peri-pubertal testicular tissue samples could be used for the organoid formation experiments. The testicular tissue samples obtained from a sample collection of the internal biobank of Department of Pathology, Karolinska University Hospital were considered normal and included in the study if no testicular pathology was reported. However, detailed information regarding previous medical treatments and/or testicular volumes of the patients included in this biobank was not available. WIDER IMPLICATIONS OF THE FINDINGS: Our observations suggest that SOX9 expression may serve as a putative indicator of TO formation, indicating a critical role of Sertoli cells in promoting organoid formation, seminiferous tubule integrity, and testicular function in pre- and peri-pubertal testicular tissue. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the Swedish Childhood Cancer Foundation (PR2019-0123; PR2022-0115; TJ2020-0023) (J.-B.S.), Finnish Cancer Society (K.J.), Finnish Foundation for Paediatric Research (K.J.), Swedish Research Council (2018-03094; 2021-02107) (J.-B.S.), and Birgitta and Carl-Axel Rydbeck's Research Grant for Paediatric Research (2020-00348; 2020-00335; 2021-00073; 2022-00317) (J.-B.S. and K.J.). Y.C. and Y.Y. received a scholarship from the Chinese Scholarship Council. J.P.A-L. was supported by a Starting Grant in Medicine and Health (2022-01467) from the Swedish Research Council. R.T.M. was supported by a UKRI Future Leaders Fellowship (MR/S017151/1). The MRC Centre for Reproductive Health was supported by an MRC Centre Grant (MR/N022556/1). The authors declare no competing interests.

In-depth analysis of transcriptomes in ovarian cortical follicles from children and adults reveals interfollicular heterogeneity.

The ovarian cortical reserve of follicles is vital for fertility. Some medical treatments are toxic to follicles, leading to premature ovarian insufficiency. Ovarian tissue cryopreservation is an established method to preserve fertility in adults and even applied in prepuberty despite unproven efficacy. Here, we analyze transcriptomes of 120 cortical follicles from children and adults for detailed comparison. We discover heterogeneity with two main types of follicles in both age groups: one with expected oocyte-granulosa profiles and another with predicted role in signaling. Transcriptional changes during growth to the secondary stage are similar overall in children and adults, but variations related to extracellular matrix, theca cells, and miRNA profiles are found. Notably, cyclophosphamide dose correlates with interferon signaling in child follicles. Additionally, morphology alone is insufficient for follicle categorization suggesting a need for additional markers. Marker genes for early follicle activation are determined. These findings will help refine follicular classification and fertility preservation techniques across critical ages.

Demographic and disease-related factors impact bone turnover and vitamin D in children with hemato-oncological diseases.

Children with hemato-oncological diseases may have significant skeletal morbidity, not only during and after treatment but also at the time of diagnosis before cancer treatment. This study was designed to evaluate the vitamin D status and circulating bone metabolic markers and their determinants in children at the time of diagnostic evaluation for hemato-oncological disease. This cross-sectional study included 165 children (91 males, median age 6.9 yr range 0.2-17.7 yr). Of them, 76 patients were diagnosed with extracranial or intracranial solid tumors, 83 with leukemia, and 6 with bone marrow failure. Bone metabolism was assessed by measuring serum 25OHD, PTH, bone alkaline phosphatase, intact N-terminal propeptide of type I procollagen, and C-terminal cross-linked telopeptide of type I collagen. Vitamin D deficiency was found in 30.9% of children. Lower 25OHD levels were associated with older age, lack of vitamin D supplementation, season outside summer, and a country of parental origin located between latitudes -45° and 45°. Children diagnosed with leukemia had lower levels of markers of bone formation and bone resorption than those who had solid tumors or bone marrow failure. In conclusion, vitamin D deficiency was observed in one-third of children with newly diagnosed cancer. Bone turnover markers were decreased in children with leukemia, possibly because of the suppression of osteoblasts and osteoclasts by leukemic cells. The identification of patients with suboptimal vitamin D status and compromised bone remodeling at cancer diagnosis may aid in the development of supportive treatment to reduce the adverse effects of cancer and its treatment.

ProFertil study protocol for the investigation of gonadotropin-releasing hormone agonists (GnRHa) during chemotherapy aiming at fertility protection of young women and teenagers with cancer in Sweden-a phase III randomised double-blinded placebo-controlled study.

BACKGROUND: Gonadotropin-releasing hormone agonists (GnRHa) cotreatment used to transiently suppress ovarian function during chemotherapy to prevent ovarian damage and preserve female fertility is used globally but efficacy is debated. Most clinical studies investigating a beneficial effect of GnRHa cotreatment on ovarian function have been small, retrospective and uncontrolled. Unblinded randomised studies on women with breast cancer have suggested a beneficial effect, but results are mixed with lack of evidence of improvement in markers of ovarian reserve. Unblinded randomised studies of women with lymphoma have not shown any benefit regarding fertility markers after long-term follow-up and no placebo-controlled study has been conducted so far. The aim of this study is to investigate if administration of GnRHa during cancer treatment can preserve fertility in young female cancer patients in a double-blind, placebo-controlled clinical trial. METHODS AND ANALYSIS: A prospective, randomised, double-blinded, placebo-controlled, phase III study including 300 subjects with breast cancer. In addition, 200 subjects with lymphoma, acute leukemias and sarcomas will be recruited. Women aged 14-42 will be randomised 1:1 to treatment with GnRHa (triptorelin) or placebo for the duration of their gonadotoxic chemotherapy. Follow-up until 5 years from end of treatment (EoT). The primary endpoint will be change in anti-Müllerian hormone (AMH) recovery at follow-up 12 months after EoT, relative to AMH levels at EoT, comparing the GnRHa group and the placebo group in women with breast cancer. ETHICS AND DISSEMINATION: This study is designed in accordance with the principles of Good Clinical Practice (ICH-GCP E6 (R2)), local regulations (ie, European Directive 2001/20/EC) and the ethical principles of the Declaration of Helsinki. Within 6 months of study completion, the results will be analysed and the study results shall be reported in the EudraCT database. STUDY REGISTRATION: The National Institutional review board in Sweden dnr:2021-03379, approval date 12 October 2021 (approved amendments 12 June 2022, dnr:2022-02924-02 and 13 December 2022, dnr:2022-05565-02). The Swedish Medical Product Agency 19 January 2022, Dnr:5.1-2021-98927 (approved amendment 4 February 2022). Manufacturing authorisation for authorised medicinal products approved 6 December 2021, Dnr:6.2.1-2020-079580. Stockholm Medical Biobank approved 22 June 2022, RBC dnr:202 253. TRIAL REGISTRATION NUMBER: NCT05328258; EudraCT number:2020-004780-71.

Reference standards for follicular density in ovarian cortex from birth to sexual maturity.

RESEARCH QUESTION: Are age-normalized reference values for human ovarian cortical follicular density adequate for tissue quality control in fertility preservation? DESIGN: Published quantitative data on the number of follicles in samples without known ovarian pathology were converted into cortical densities to create reference values. Next, a sample cohort of 126 girls (age 1-24 years, mean ± SD 11 ± 6) with cancer, severe haematological disease or Turner syndrome were used to calculate Z-scores for cortical follicular density based on the reference values. RESULTS: No difference was observed between Z-scores in samples from untreated patients (0.3 ± 3.5, n = 30) and patients treated with (0.5 ± 2.9, n = 48) and without (0.1 ± 1.3, n = 6) alkylating chemotherapy. Z-scores were not correlated with increasing cumulative exposure to cytostatics. Nevertheless, Z-scores in young treated patients (0-2 years -2.1 ± 3.1, n = 10, P = 0.04) were significantly lower than Z-scores in older treated patients (11-19 years, 2 ± 1.9, n = 15). Samples from patients with Turner syndrome differed significantly from samples from untreated patients (-5.2 ± 5.1, n = 24, P = 0.003), and a Z-score of -1.7 was identified as a cut-off showing good diagnostic value for identification of patients with Turner syndrome with reduced ovarian reserve. When this cut-off was applied to other patients, analysis showed that those with indications for reduced ovarian reserve (n = 15) were significantly younger (5.9 ± 4.2 versus 10.7 ± 5.9 years, P = 0.004) and, when untreated, more often had non-malignant haematologic diseases compared with those with normal ovarian reserve (n = 24, 100% versus 19%, P = 0.009). CONCLUSION: Z-scores allow the estimation of genetic- and treatment-related effects on follicular density in cortical tissue from young patients stored for fertility preservation. Understanding the quality of cryopreserved tissue facilitates its use during patient counselling. More research is needed regarding the cytostatic effects found in this study.

The human cathelicidin hCAP-18 in serum of children with haemato-oncological diseases.

The human cathelicidin hCAP-18 (pro-LL-37) is the pro-protein of the antimicrobial peptide LL-37. hCAP-18 can be produced by many different cell types; bone marrow neutrophil precursors are the main source of hCAP-18 in the circulation. Neutrophil count is used as a marker for myelopoiesis but does not always reflect neutrophil production in the bone marrow, and thus additional markers are needed. In this study, we established the reference interval of serum hCAP-18 level in healthy children and compared serum hCAP-18 levels between different diagnostic groups of children with haemato-oncological diseases, at diagnosis. We found that children with diseases that impair myelopoiesis, such as acute leukaemia, aplastic anaemia, or myelodysplastic syndrome, presented with low hCAP-18 levels, whereas patients with non-haematological malignancies displayed serum hCAP-18 levels in the same range as healthy children. Children with chronic myeloid leukaemia presented with high circulating levels of hCAP-18, probably reflecting the high number of all differentiation stages of myeloid cells. We suggest that analysis of serum hCAP-18 provides additional information regarding myelopoiesis in children with haemato-oncological diseases, which may have future implications in assessment of myelopoiesis in clinical management.

Motor performance after treatment of pilocytic astrocytoma in the posterior fossa in childhood.

BACKGROUND: Pilocytic astrocytoma is the most common brain tumour type in childhood located in the posterior fossa, and treated mainly with surgery. These tumours have low mortality, but knowledge concerning its long-term outcome is sparse. AIMS: The aim was to investigate if patients treated for pilocytic astrocytoma in the posterior fossa had motor complications, including balance, motor and process skills. METHODS AND RESULTS: This descriptive single-centre study includes eight children and 12 adults, treated for pilocytic astrocytoma as children. Motor performance was investigated with Bruininks-Oseretsky Test of Motor Proficiency, Second Edition, and dynamic balance with the mini-balance evaluation systems test. Physiological cost index, six-minute walk test, hand grip strength and assessment of motor and process skills were also evaluated. Ten patients reported motor difficulties, mainly from the upper limbs. The motor performance test showed results within normal limits except for manual dexterity, which was significantly below mean (p = .008). In the dynamic balance test patients had significantly lower results compared with controls (p = .036). Physiological cost index, six-minute walk tests and hand grip strength showed results within normal limits. In the Assessment of Motor and Process Skills, patients over 16 years had significantly lower results compared with test norms for motor activities of daily living (ADL) and 30% of all patients scored below the cut-off level for difficulties with motor skills. CONCLUSIONS: Motor performance for patients treated for pilocytic astrocytoma in the posterior fossa in childhood is satisfactory but some patients display difficulties with balance, manual dexterity and ADL motor skills. Thus, it is important to identify those in need of motor follow-up and training.

Cognitive, language, and school performance in children and young adults treated for low-grade astrocytoma in the posterior fossa in childhood.

BACKGROUND: Pilocytic astrocytoma is the most common brain tumour type in childhood located in the posterior fossa, and treated mainly with surgery. These tumours have low mortality, but knowledge concerning its long-term outcome is sparse. AIM: The aim of this study was to investigate whether children treated for pilocytic astrocytoma in the posterior fossa had late complications affecting cognition, language and learning. METHODS: This descriptive single-centre study includes eight children and 12 adults treated as children for pilocytic astrocytoma in the posterior fossa, with a mean follow-up time of 12.4 (range 5-19) years. Well-established tests of intelligence, executive, language and academic function were used. RESULTS: Intelligence tests showed average results compared with norms. Five patients scored <-1 SD (70-84) and 3 low average (85-92) on full scale IQ. The patients scored average on subtests regarding executive function, except for significantly lower results in inhibition/switching (p = .004). In Rey complex figure test half of the patients scored below -1 SD. Language tests were normal except for significantly lower results in naming ability (p = .049) and in inference (p = .046). In academic tests, results were average, except for significantly lower results in reading speed (p = .024). Patients with learning difficulties performed worse in the tests. CONCLUSIONS: The patients' functional outcome was favourable but, a not-negligible part of the patients displayed neurocognitive difficulties as revealed by extensive neuro-cognitive and academic testing. Thus, it is important to identify those in need of more thorough cognitive and pedagogic follow-up programmes, including school interventions.

Prevalence of and factors influencing vitamin D deficiency in paediatric patients diagnosed with cancer at northern latitudes.

AIM: To investigate the prevalence of vitamin D deficiency among children with non-haematological malignancies and to explore possible causes of low vitamin D levels among these patients. METHODS: We performed a cross-sectional study of 458 children diagnosed with solid tumours, brain tumours, non-Hodgkin lymphoma or Hodgkin disease at the University Children's Hospital, Uppsala, Sweden. Serum 25-hydroxyvitamin D and parathyroid hormone levels were measured in samples taken at the time of cancer diagnosis and related to clinical data. Vitamin D deficiency was defined as a 25-hydroxyvitamin D level below 50 nmol/L. RESULTS: The prevalence rate of vitamin D deficiency among children with non-haematological malignancies was 41%. There was no association between sex or diagnosis and vitamin D status. Vitamin D deficiency was more common among school children than preschool children (51% vs. 24%). Older age, season outside summer, and a more recent calendar year were significant predictors of lower 25-hydroxyvitamin D. There was a significant, albeit weak, negative correlation between 25-hydroxyvitamin D and parathyroid hormone. CONCLUSION: Vitamin D deficiency is common among children diagnosed with cancer, particularly among school-aged children diagnosed outside summer. The prevalence appears to be increasing, underlining the need for adequate replacement of vitamin D in these patients.

Genetic and functional insights into CDA-I prevalence and pathogenesis.

BACKGROUND: Congenital dyserythropoietic anaemia type I (CDA-I) is a hereditary anaemia caused by biallelic mutations in the widely expressed genes CDAN1 and C15orf41. Little is understood about either protein and it is unclear in which cellular pathways they participate. METHODS: Genetic analysis of a cohort of patients with CDA-I identifies novel pathogenic variants in both known causative genes. We analyse the mutation distribution and the predicted structural positioning of amino acids affected in Codanin-1, the protein encoded by CDAN1. Using western blotting, immunoprecipitation and immunofluorescence, we determine the effect of particular mutations on both proteins and interrogate protein interaction, stability and subcellular localisation. RESULTS: We identify six novel CDAN1 mutations and one novel mutation in C15orf41 and uncover evidence of further genetic heterogeneity in CDA-I. Additionally, population genetics suggests that CDA-I is more common than currently predicted. Mutations are enriched in six clusters in Codanin-1 and tend to affect buried residues. Many missense and in-frame mutations do not destabilise the entire protein. Rather C15orf41 relies on Codanin-1 for stability and both proteins, which are enriched in the nucleolus, interact to form an obligate complex in cells. CONCLUSION: Stability and interaction data suggest that C15orf41 may be the key determinant of CDA-I and offer insight into the mechanism underlying this disease. Both proteins share a common pathway likely to be present in a wide variety of cell types; however, nucleolar enrichment may provide a clue as to the erythroid specific nature of CDA-I. The surprisingly high predicted incidence of CDA-I suggests that better ascertainment would lead to improved patient care.

Vitamin D status in children with leukemia, its predictors, and association with outcome.

BACKGROUND: Children and adolescents with leukemia are potentially at high risk of vitamin D inadequacy, which may have clinical relevance for skeletal morbidity, infections, and cancer outcome. This study aimed to evaluate vitamin D status at the time of diagnosis to investigate its predictors and association with overall survival in children with leukemia. PROCEDURE: We included all 295 children and adolescents diagnosed with leukemia at our institution between 1990 and 2016 who had available serum sample from the time of diagnosis. We analyzed serum 25-hydroxyvitamin D and parathyroid hormone levels and correlated them with clinical data. RESULTS: The 25-hydroxyvitamin D level was deficient (< 25 nmol/L), insufficient (25-50 nmol/L), sufficient (50-75 nmol/L), and optimal (> 75 nmol/L) in 6.4%, 26.8%, 39.7%, and 27.1% of the children, respectively. Older age and a more recent time of sampling (calendar year) predicted lower 25-hydroxyvitamin D level. In preschool children (age ≤6 years), lower 25-hydroxyvitamin D level was also associated with acute myeloid leukemia, and a 25-hydroxyvitamin D level < 50 nmol/L was associated with inferior overall survival. In school-aged children (age > 6 years), the 25-hydroxyvitamin D level showed significant seasonal variation. CONCLUSION: It remains unclear whether vitamin D supplementation in pediatric leukemia patients will improve outcome.

Clinical characteristics, long-term complications and health-related quality of life (HRQoL) in children and young adults treated for low-grade astrocytoma in the posterior fossa in childhood.

INTRODUCTION: Pilocytic astrocytoma is the most common brain tumour in childhood but knowledge concerning its long-term outcome is sparse. The aim of the study was to investigate if children treated for low-grade pilocytic astrocytoma in the posterior fossa had complications affecting physical and psychological health, cognitive functions, learning difficulties and HRQoL. METHODS: A descriptive single-centre study, where 22 children and young adults out of 27 eligible patients (81%) treated for pilocytic astrocytoma, with a mean follow-up time of 12.4 years (5-19 years) participated (14 adults, two by telephone interviews and eight children). The study included a review of medical records, an interview, neurological investigation, screening tools for psychiatric symptoms (Beck Depression and Anxiety Inventories and Beck Youth Inventory Scales) and HRQoL measures (RAND-36). RESULTS: Motor complications were most common, reported in 12 patients and mainly affecting fine-motor skills. Seven patients reported cognitive difficulties affecting performance in school. Educational support was given in the period immediately after treatment but not after primary school. None had elevated levels of psychiatric symptoms and the level of HRQoL as well as their psychosocial and educational situation was in correspondence with Swedish norms. The HRQoL score for vitality (VT) almost reached statistical significance. CONCLUSIONS: The long-term functional outcome for children treated for low-grade astrocytoma is favourable. However, some patients report neurological complications and learning difficulties, which are unmet in school. Therefore, there is a need to identify those who need more thorough medical and cognitive follow-up programmes including interventions in school.

Exercise capacity in young adults after hematopoietic cell transplantation in childhood.

A symptom-limited incremental cycle ergometer test was performed in 17 young adult patients treated with hematopoietic cell transplantation and total body irradiation for hematologic malignancies during childhood. These 17 young adult patients were compared with 17 sex- and age-matched healthy control subjects. Assessments of pulmonary function, cardiac function, body composition, and levels of growth hormone were made. The median follow-up was 17.7 years. Patients achieved 63.2% of the predicted peak workload, whereas controls achieved 96.1% (P < .001). All patients, but only 1 control, failed to achieve a peak workload >80% (P < .001). Fat-free mass was significantly lower (43.5 vs 57.6 kg, P < .001) and fat mass percentage was significantly higher (31.8% vs 24.2%, P = .011) in the patients. The peak workload adjusted for fat-free mass was significantly lower in the patients (3.3 vs 4.3, P < .001). In the patients, peak workload correlated significantly with total lung capacity (r = .54, P = .025). In summary, long-term survivors have significantly decreased exercise capacity compared with healthy individuals. Together with their altered body composition, this may predispose them to cardiovascular disease.

Successful treatment with placenta-derived decidual stromal cells in a pediatric patient with life-threatening acute gastrointestinal graft-versus-host disease.

Severe aGvHD is a life-threatening complication after allogeneic HSCT. The GI tract is considered to play a key role in aGvHD, where the disease process can start and is one of the major target organs. Here, we present a case of a one-year-old child with a life-threatening GI-aGvHD stage IV, post-HSCT, resistant to steroids and MMF for 4 weeks. He was successfully treated with placenta-derived DSC.

Self-reported quality of life in long-term survivors of childhood lymphoblastic malignancy treated with hematopoietic stem cell transplantation versus conventional therapy.

BACKGROUND: Chronic health conditions are known to be both abundant and severe after pediatric hematopoietic stem cell transplantation (SCT). The present objective was to investigate the impact of disease and treatment on individual QoL and health-related quality of life (HRQoL) in long-term survivors of childhood lymphoblastic malignancy treated with conventional therapy versus SCT. PROCEDURE: Survivors of lymphoblastic malignancy treated with (n = 18) or without (n = 52) SCT were recruited a median follow-up time of 18 and 14 years, respectively. The indication for SCT was relapsed disease in 17 of 18 cases. Autologous stem cells were used in 15 cases. Total body irradiation (TBI) was included in the conditioning regimen for all SCT patients. A cross-sectional study was conducted using two validated instruments: SEIQoL-DW (individual QoL) and SF-36 (HRQoL). Content analysis was used to analyze SEIQoL-DW and an overall QoL index score was calculated. Two multiple linear regression analyses were performed to detect factors influencing outcomes. RESULTS: Poorer ratings of overall QoL and more negative consequences related to physical dysfunctions were shown in the SCT group. The findings indicate that being unemployed or on sick leave are associated with a decline in HRQoL and individual QoL rather than SCT, cranial radiation therapy, present age, or sex. CONCLUSION: In this small sample of long-term survivors of SCT, QoL seems reasonably good and similar to that of those having received conventional therapy. However, managing an employment must be acknowledged as an important part of life that has a great impact on QoL.

Comparison of primary human cytotoxic T-cell and natural killer cell responses reveal similar molecular requirements for lytic granule exocytosis but differences in cytokine production.

Cytotoxic lymphocytes, encompassing cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, kill pathogen-infected, neoplastic, or certain hematopoietic cells through the release of perforin-containing lytic granules. In the present study, we first performed probability-state modeling of differentiation and lytic granule markers on CD8(+) T cells to enable the comparison of bona fide CTLs with NK cells. Analysis identified CD57(bright) expression as a reliable phenotype of granule marker-containing CTLs. We then compared CD3(+)CD8(+)CD57(bright) CTLs with NK cells. Healthy adult peripheral blood CD3(+)CD8(+)CD57(bright) CTLs expressed more granzyme B but less perforin than CD3(-)CD56(dim) NK cells. On stimulation, such CTLs degranulated more readily than other T-cell subsets, but had a propensity to degranulate that was similar to NK cells. Remarkably, the CTLs produced cytokines more rapidly and with greater frequency than NK cells. In patients with biallelic mutations in UNC13D, STX11, or STXBP2 associated with familial hemophagocytic lymphohistiocytosis, CTL and NK cell degranulation were similarly impaired. Therefore, cytotoxic lymphocyte subsets have similar requirements for Munc13-4, syntaxin-11, and Munc18-2 in lytic granule exocytosis. The present results provide a detailed comparison of human CD3(+)CD8(+)CD57(bright) CTLs and NK cells and suggest that analysis of CD57(bright) CTL function may prove useful in the diagnosis of primary immunodeficiencies including familial hemophagocytic lymphohistiocytosis.

Risk factors for cardiovascular disease are increased in young adults treated with stem cell transplantation during childhood.

We measured risk factors for CVD in 18 patients at a median of 18.2 yr after SCT and in sex and age-matched controls. Three patients (17%), but none of the controls, met the criteria for the MetS (p = 0.25). In the patients, we found higher levels of triglycerides (0.94 vs. 0.62 mm, p = 0.019), total cholesterol (5.1 vs. 4.0 mm, p = 0.017), LDL (3.4 vs. 2.6 mm, p = 0.019), apolipoprotein B (1.04 vs. 0.74 g/L, p = 0.004), apolipoprotein B/A1 ratio (0.7 vs. 0.5, p = 0.026), and lower levels of adiponectin (4.9 vs. 7.5 mg/L, p = 0.008) than in the controls. The patients had a lower GHmax (9 vs. 20.7 mU/L, p = 0.002). GHmax was significantly correlated inversely with triglycerides (r = -0.64, p = 0.008), total cholesterol (r = -0.61, p = 0.011), apolipoprotein B (r = -0.60, p = 0.014), and apolipoprotein B/A1 ratio (r = -0.66, p = 0.005). We recorded a significantly thicker carotid intima layer among the patients than among matched controls (0.15 vs. 0.13 mm, p = 0.034). The level of adiponectin correlated inversely with carotid intima thickness (r = -0.55, p = 0.023). After SCT in childhood, long-term survivors may be at risk of developing premature CVD.

A longitudinal study of pulmonary function after stem cell transplantation, from childhood to young adulthood.

BACKGROUND: Impairment of pulmonary function after stem cell transplantation (SCT) in childhood has been reported before. However, long-term longitudinal studies are scarce. PROCEDURE: We measured lung volumes and performed dynamic spirometry serially in 18 patients after SCT. At the last investigation, a median of 18.2 years after SCT, the patients were compared with 18 matched controls. The diffusing capacity (DLCO) was only compared cross-sectionally. RESULTS: There was a significant increase in the prevalence of restrictive lung disease (RLD, total lung capacity <80% of that predicted) from 7% (1/14) before SCT to 28% (5/18) 5 years after SCT, and 61% (11/18) a median of 18.2 years after SCT (P = 0.002). In comparison, none of the controls had RLD (61% vs. 0%, P = 0.001). Before SCT, no patient had obstructive lung disease (OLD, forced expiratory volume in 1 sec/vital capacity <70). OLD was found in one of 18 patients (6%) 5 years after SCT but in none of the patients a median of 18.2 years after SCT. Three of the controls had OLD (P = 0.25). Eleven patients had diffusion impairment (DLCO <80% of that predicted), as opposed to none of the controls (P = 0.001). The DLCO corrected for alveolar volume was decreased in only two patients. CONCLUSION: We documented an increase in the prevalence of RLD, but not of OLD, after SCT. At the last investigation, only two patients had diffusion impairment after correction for alveolar volume.

Both CD4+ FoxP3+ and CD4+ FoxP3- T cells from patients with B-cell malignancy express cytolytic markers and kill autologous leukaemic B cells in vitro.

Cytotoxic CD4(+) T cells have been found in patients with chronic lymphocytic leukaemia (CLL) and seem to be involved in the regulation of malignant B cells. The CD4(+) T regulatory cells (Tregs) can regulate various immune cells, including B cells, by inducing their apoptosis. Hence, different subgroups of CD4(+) T cells may be involved in the regulation of malignant B cells. In this study, the cytotoxic phenotype and function of various CD4(+) T-cell subgroups were investigated in patients with B-cell malignancies. Peripheral blood was collected from patients with CLL, various B-cell lymphomas, healthy adult donors, children with precursor B-cell acute lymphoblastic leukaemia (pre-B ALL) and from healthy children. CD4(+) T cells (CD3(+) CD4(+) FoxP3(-)), Tregs (CD3(+) CD4(+) CD127(low) FoxP3(+)) and CD127(high) FoxP3(+) T cells (CD3(+) CD4(+) CD127(high) FoxP3(+)) were analysed for their expression of the cytolytic markers CD107a and Fas ligand. Patients with CLL had increased CD107a expression on all tested T-cell subgroups compared with healthy donors. Similar results were found in patients with B-cell lymphomas whereas the CD107a expression in children with pre-B ALL was no different from that in healthy controls. Fas ligand expression was similar between patient cells and cells of healthy donors. CD4(+) T cells and Tregs from patients with CLL and healthy donors were subsequently purified and cultured in vitro with autologous B cells. Both subgroups lysed B cells and killing was confirmed by granzyme ELISAs. In conclusion, cytotoxic populations of CD4(+) T cells, including Tregs, are present in patients with B-cell malignancy and may be an important factor in immune-related disease control.

Glomerular and tubular function in young adults treated with stem-cell transplantation in childhood.

We evaluated renal function at a median follow-up of 18 (range 10.3-22.1) years after total body irradiation in 18 patients treated with stem-cell transplantation (SCT) (autologous SCT in 15 and allogeneic SCT in three) for hematologic malignancies and compared them with 18 healthy controls. No patient had chronic graft-versus-host disease. We found no difference in glomerular filtration rate estimated from cystatin C (105 vs 111 ml/min/1.73 m(2), p = 0.28). Patients had higher albumin excretion (0.8 vs 0.4 mg/mmol, p = 0.001), but no patient had overt albuminuria (>200 mg/L). Patients had higher diastolic blood pressure (74 vs 67 mmHg, p = 0.003). Two patients (11%) had hypertension. Patients had lower tubular reabsorption of phosphate (0.78 vs 0.91 mmol/L, p = 0.014) and higher excretion of alpha-1-microglobulin (AMG/urine creatinine, 0.4 vs 0.25 mg/mmol, p = 0.038), which correlated with time after SCT (r = 0.6, p = 0.01). We found no difference in fractional excretion (FE) of other electrolytes, amino acid excretion, or urine osmolality. We conclude that renal function was relatively well preserved at a median follow-up of 18 years after childhood SCT. The higher albumin excretion in our patients is of concern, as is the association between excretion of AMG and time after SCT, suggesting that both glomerular and tubular function may deteriorate further.