Peripheral blood lymphocyte immunophenotyping (TBNK) - a comparison of BD FACSCanto II and BD FACSLyric flow cytometry analysers.

Introduction: Flow cytometry immunophenotyping is a common laboratory technique for evaluating lymphocyte subpopulations. Its result remains an important diagnostic tool in various medical fields. Cytometric tests are performed in many laboratories, making the comparability between different devices using the same method an important aspect. We aimed to compare the results of lymphocyte immunophenotyping (lymphocytes B, T, Th and Tc, NK cells) between two different flow cytometers. Material and methods: The study included 93 patients of the Children's Teaching Hospital of the Medical University of Warsaw and 9 Multi-Check control results. The method of lymphocyte subpopulation assessment was based on fluorescent flow cytometry immunophenotyping, using a BD Multitest 6-color TBNK kit (Becton Dickinson). We compared BD FACSCanto II and BD FACSLyric analysers (Becton Dickinson). For data analysis, we used Spearman's rank correlation, Bland-Altman plot and Passing-Bablok regression. Results: Spearman's rank correlation showed a strong interrelation for all analysed parameters (0.808-0.985). In the Passing-Bablok regression analysis, all examined parameters showed linear dependence with the slope values close to 1 (0.940-1.134). Bland-Altman coefficient values were within the range of 2.94-8.62% with half of them being above 5% (T, Tc, Th, B, NKT absolute values and B percentage values). Conclusions: The results from both cytometers can be considered equivalent, but it should be noted that one of the statistical methods showed some deviations, presumably primarily due to the evaluators' different gating techniques. The training of specialists performing these tests requires more attention.

Hyperthermia Enhances Adeno-Associated Virus Vector Transduction Efficiency in Melanoma Cells.

Gene therapy perfectly fits in the current needs of medicine for patients with melanoma. One of the major challenges of gene therapy is to increase gene transfer. The role of hyperthermia in the improvement of AAV (adeno-associated virus) transduction efficiency has been indicated. The aim of the present study was to assess the transduction efficacy of melanoma cell lines (A375, G-361, and SK-MEL-1) with the use of the rAAV/DJ mosaic vector under hyperthermia conditions. The analysis of changes in the transduction efficacy and expression of HSPs (heat shock proteins) and receptors for AAV was performed. The transduction was performed at 37 °C and at 43 °C (1 h). Hyperthermia enhanced gene transfer in all the tested cell lines. The most efficient transducing cell line under hyperthermia was A375 (increase by 17%). G361 and SK-MEL-1 cells showed an increase of 7%. The changes in the expression of the AAV receptors and HSPs after hyperthermia were observed. A key role in the improvement of gene transfer may be played by AAVR, HSPB1, HSP6, DNAJC4, HSPD1, HSPA8, HSPA9, HSP90AB1, and AHSA1. This study showed the possibility of the use of hyperthermia as a factor enabling the stimulation of cell transduction with rAAV vectors, thereby providing tools for the improvement in the efficacy of gene therapy based on rAAV.

Data-driven case fatality rate estimation for the primary lineage of SARS-CoV-2 in Poland.

After more than one and a half year since the COVID-19 pandemics outbreak the scientific world is constantly trying to understand its dynamics. In this paper of the case fatality rates (CFR) for COVID-19 we study the historic data regarding mortality in Poland during the first six months of pandemic, when no SARS-CoV-2 variants of concern were present among infected. To this end, we apply competing risk models to perform both uni- and multivariate analyses on specific subpopulations selected by different factors including the key indicators: age, sex, hospitalization. The study explores the case fatality rate to find out its decreasing trend in time. Furthermore, we describe the differences in mortality among hospitalized and other cases indicating a sudden increase of mortality among hospitalized cases at the end of the 2020 spring season. Exploratory and multivariate analysis revealed the real impact of each variable and besides the expected factors indicating increased mortality (age, comorbidities) we track more non-obvious indicators. Recent medical care as well as the identification of the source contact, independently of the comorbidities, significantly impact an individual mortality risk. As a result, the study provides a twofold insight into the COVID-19 mortality in Poland. On one hand we explore mortality in different groups with respect to different variables, on the other we indicate novel factors that may be crucial in reducing mortality. The later can be coped, e.g. by more efficient contact tracing and proper organization and management of the health care system to accompany those who need medical care independently of comorbidities or COVID-19 infection.

Molecular Mechanisms of Neutrophil Extracellular Trap (NETs) Degradation.

Although many studies have been exploring the mechanisms driving NETs formation, much less attention has been paid to the degradation and elimination of these structures. The NETs clearance and the effective removal of extracellular DNA, enzymatic proteins (neutrophil elastase, proteinase 3, myeloperoxidase) or histones are necessary to maintain tissue homeostasis, to prevent inflammation and to avoid the presentation of self-antigens. The persistence and overabundance of DNA fibers in the circulation and tissues may have dramatic consequences for a host leading to the development of various systemic and local damage. NETs are cleaved by a concerted action of extracellular and secreted deoxyribonucleases (DNases) followed by intracellular degradation by macrophages. NETs accumulation depends on the ability of DNase I and DNAse II to hydrolyze DNA. Furthermore, the macrophages actively engulf NETs and this event is facilitated by the preprocessing of NETs by DNase I. The purpose of this review is to present and discuss the current knowledge about the mechanisms of NETs degradation and its role in the pathogenesis of thrombosis, autoimmune diseases, cancer and severe infections, as well as to discuss the possibilities for potential therapeutic interventions. Several anti-NETs approaches had therapeutic effects in animal models of cancer and autoimmune diseases; nevertheless, the development of new drugs for patients needs further study for an effective development of clinical compounds that are able to target NETs.

Influence of blood sample storage and different types of anticoagulants on results of NK cytotoxicity tests.

Introduction: Natural killer (NK) cells are important players in the human immune response. Impaired NK function may lead to serious, life-threatening conditions. Defects may be consequences of genetic mutations or results of secondary factors such as infections, malignancies and autoimmune diseases. The cytotoxicity test is very useful, but its accessibility is limited to special immunological laboratories. Blood samples are often transported to remote centers, which takes time and requires special conditions.The aim of this study was to compare cytotoxicity assay results between samples preserved with three different anticoagulants to standardize the diagnostic procedure. Material and methods: Peripheral blood from healthy donors was taken with three anticoagulants: heparin, K2EDTA and citrate. Peripheral blood mononuclear cells (PBMC) were isolated and tested directly after blood drawing and after 24-hour storage. Cytotoxic abilities of NK cells were tested in 4 h co-culture with K562. NK cytotoxicity was measured by flow cytometry. Results: In most cases of analyzed healthy donors, cytotoxicity results were similar regardless of type of anticoagulant. However, the highest mean values were obtained in samples with citrate. There was a significant decrease in cytotoxicity after 24 hours of storage of the whole blood at ambient temperature. The mean drop in cytotoxicity results was substantial for all anticoagulants: 76% for heparin, 67% for citrate and 70% for EDTA. Conclusions: Results of spontaneous NK cytotoxicity seem to be affected by the anticoagulants used for blood protection. Commercial instant cytotoxicity testing and delayed analysis after blood storage gave the highest results in blood with sodium citrate.

Iron excess affects release of neutrophil extracellular traps and reactive oxygen species but does not influence other functions of neutrophils.

Neutrophils apply several antimicrobial strategies including degranulation, phagocytosis, the generation of reactive oxygen species (ROS) and the release of neutrophil extracellular traps (NETs) to fight pathogens. Iron is considered to be an invaluable constituent of host immune defense and plays a dual role in immunity. It is a well-known component of antimicrobial proteins and is a necessary microelement for pathogen survival. The aim of this study was to broaden the knowledge regarding the impact of iron on the function of neutrophils. Neutrophils from healthy blood donors and patients with mild iron-deficiency anemia and HL-60 cells differentiated toward granulocyte-like cells were incubated with Fe2+ , Fe3+ or holo-transferrin (holo-Tf). Moreover, we isolated murine neutrophils of HFE gene knockout (KO) mice and mice fed iron-deficient, iron-equivalent and high-iron diets. We analyzed the release of NETs, phagocytosis, degranulation of azurophilic granules, ROS release, bactericidal activity of granulocytes against Escherichia coli and neutrophil elastase (NE) activity. We show that holo-Tf inhibits the release of NETs stimulated by phorbol 12-myristate 13-acetate by inhibiting NE activity. Studies performed in mice models reveal that iron overload inhibits the release of NETs and ROS production in neutrophils isolated from HFE KO mice and mice fed a high-iron diet. No impact of a low-iron diet on neutrophil phagocytosis, ROS production or release of NETs was observed. Our study underscores the physiological significance of iron in neutrophil function, specifically in the release of NETs.

Ultrasensitive voltammetric detection of SARS-CoV-2 in clinical samples.

In every pandemic, it is critical to test as many people as possible and keep track of the number of new cases of infection. Therefore, there is a need for novel, fast and unambiguous testing methods. In this study, we designed a sandwich-type voltammetric immunosensor based on unlabeled- and labeled with a redox probe antibodies against virus spike protein for fast and ultrasensitive detection of SARS-CoV-2. The process of the preparation of the sensor layer included chemisorption of cysteamine layer and covalent anchoring of antibody specific for the S1 subunit of the S protein. The source of the voltametric signal was the antibody labeled with the redox probe, which was introduced onto biosensor surface only after the recognition of the virus. This easy-to-handle immunosensor was characterized by a wide analytical range (2.0·10-7 to 0.20 mg·L-1) and low detection limit (8.0·10-8 mg·L-1 ≡ 0.08 pg·mL-1 ≡ 4 virions·µL-1). The utility of the designed device was also evidenced by the detection of SARS-CoV-2 in the clinical samples. Moreover, the main advantage and a huge novelty of the developed device, compared to those already existing, is the moment of generating the analytical signal of the redox probe that appears only after the virus recognition. Thus, our diagnostic innovation may considerably contribute to controlling the COVID-19 pandemic. The as-developed immunosensor may well offer a novel alternative approach for viral detection that could complement or even replace the existing methods.

The Impact of Cytokines on Neutrophils' Phagocytosis and NET Formation during Sepsis-A Review.

Sepsis is an overwhelming inflammatory response to infection, resulting in multiple-organ injury. Neutrophils are crucial immune cells involved in innate response to pathogens and their migration and effector functions, such as phagocytosis and neutrophil extracellular trap (NET) formation, are dependent on cytokine presence and their concentration. In the course of sepsis, recruitment and migration of neutrophils to infectious foci gradually becomes impaired, thus leading to loss of a crucial arm of the innate immune response to infection. Our review briefly describes the sepsis course, the importance of neutrophils during sepsis, and explains dependence between cytokines and their activation. Moreover, we, for the first time, summarize the impact of cytokines on phagocytosis and NET formation. We highlight and discuss the importance of cytokines in modulation of both processes and emphasize the direction of further investigations.

Lack of Functional P110δ Affects Expression of Activation Marker CD80 but Does Not Influence Functions of Neutrophils.

Neutrophils are specialized immune cells that are essential constituents of the innate immune response. They defend the organism against pathogens through various mechanisms. It was reported that phosphatidylinositols are key players in neutrophil functions, especially in the activity of class-I phosphoinositide 3-kinases (PI3Ks). P110δ, one of the PI3K subunits, is mostly expressed in immune cells, and its activity plays an important role in inflammatory responses. The aim of this study was to investigate the role of p110δ in neutrophil antimicrobial functions, activation status and cytokine production. To this end, we used bone marrow and splenic neutrophils isolated from a murine model expressing catalytically inactive p110δD910A/D910A. The level of phagocytosis and degranulation, the expressions of activation markers and cytokine production were determined by flow cytometry. ROS generation and NET release were assessed by fluorometry and fluorescent microscopy. We observed a significantly higher percentage of CD80-positive cells among the splenic granulocytes and found granulocytes subpopulations of differing phenotypes between WT and p110δD910A/D910A mice by multiparametric tSNE analysis. Moreover, we detected some differences in the expressions of activation markers, intracellular production of cytokines and bacterial killing. However, we did not observe any alterations in the selected neutrophil functions in p110δ mutant mice. Altogether, our data suggest that the catalytic p110 subunit(s), other than p110δ, is a key player in most neutrophil functions in mice. A follow-up study to correlate these in vitro results with in vivo observations is highly recommended.

Serum and urine periostin and cytokeratin-18 in children with congenital obstructive nephropathy.

Congenital obstructive nephropathy (CON) is one of the most common causes of chronic kidney disease in children. The aim of the study was to investigate serum and urine periostin and cytokeratin-18 (CK-18) in children with CON in relation to CON etiology, treatment, and kidney injury. We evaluated 81 children with CON secondary to ureteropelvic junction obstruction (UPJO), ureterovesical junction obstruction (UVJO), posterior urethral valves (PUV) and 60 controls. Neither biomarker demonstrated any relation to CON etiology. However, all patients showed significantly higher urine periostin (uPeriostin) and uPeriostin/Cr levels than the controls. Also, UVJO patients showed higher sCK-18 and uCK-18/Cr levels, and PUV patients showed higher uCK-18/Cr levels than the controls. Neither biomarker was found to have any relation to CON treatment. However, conservatively treated children and those before and after surgery showed significantly higher uPeriostin and uPeriostin/Cr levels than the controls. uPeriostin strongly correlated with differential renal function (DRF) < 40%. The ROC analysis demonstrated the best area under the curve (AUC) for uPeriostin (0.831) and uPeriostin/Cr (0.768), and low for sPeriostin (0.656) and uCK-18 (0.615) for detecting renal injury. In conclusion, although serum and urine periostin and CK-18 did not display any relation to etiology or the type of CON treatment, uPeriostin seems to be a useful tool for detecting renal injury in children with CON, especially due to its strong negative correlation with DRF < 40%.

Recurrent assessment of lymphocyte subsets in 32 patients with multisystem inflammatory syndrome in children (MIS-C).

BACKGROUND: Lymphopenia is a hallmark of multisystem inflammatory syndrome in children (MIS-C). We aimed to characterize lymphocyte subsets' shifts and their correlations with other severity markers of MIS-C. METHODS: In this prospective cross-sectional study, we performed peripheral lymphocyte phenotyping in 32 patients with MIS-C. We analyzed lymphocyte subsets at three time points of the disease: the acute (A), convalescent (B), and recovery (C) phases. Based on age-normalized lymphocyte counts, we distinguished two groups of patients: "the mild" (higher lymphocyte counts) and "the severe" (lower lymphocyte counts). In addition, we examined differences between these groups regarding other severity markers. RESULTS: In phase A, 84% of children had lymphopenia. Decreased absolute counts of CD3, CD4, and CD8 cells were observed in, respectively, 88%, 72%, and 84% of patients. The natural killer cells were decreased in 63% and CD19 in 59% of children. "The severe" group had significantly higher procalcitonin and troponin I levels and lower platelets and albumin. Moreover, "the severe" group had hypotension more frequently (73% vs. 20%, p = .008). In phase B, all lymphocyte counts increased, and 32% of children had lymphocytosis. The increase of CD3, CD4, and CD8 counts correlated with some laboratory severity markers (hemoglobin, procalcitonin, D-dimer, lactate dehydrogenase, N-terminal prohormone of brain natriuretic peptide, albumin), but not with steroid use. In phase C, most children had normal lymphocyte counts. CONCLUSIONS: Substantial shifts in lymphocyte counts during MIS-C apply most to T lymphocytes and correlate with the disease severity markers, particularly hypotension prevalence. A proportion of children with MIS-C develops transient lymphocytosis during convalescence.

Nitric oxide and peroxynitrite trigger and enhance release of neutrophil extracellular traps.

Despite great interest, the mechanism of neutrophil extracellular traps (NETs) release is not fully understood and some aspects of this process, e.g. the role of reactive nitrogen species (RNS), still remain unclear. Therefore, our aim was to investigate the mechanisms underlying RNS-induced formation of NETs and contribution of RNS to NETs release triggered by various physiological and synthetic stimuli. The involvement of RNS in NETs formation was studied in primary human neutrophils and differentiated human promyelocytic leukemia cells (HL-60 cells). RNS (peroxynitrite and nitric oxide) efficiently induced NETs release and potentiated NETs-inducing properties of platelet activating factor and lipopolysaccharide. RNS-induced NETs formation was independent of autophagy and histone citrullination, but dependent on the activity of phosphoinositide 3-kinases (PI3K) and myeloperoxidase, as well as selective degradation of histones H2A and H2B by neutrophil elastase. Additionally, NADPH oxidase activity was required to release NETs upon stimulation with NO, as shown in NADPH-deficient neutrophils isolated from patients with chronic granulomatous disease. The role of RNS was further supported by increased RNS synthesis upon stimulation of NETs release with phorbol 12-myristate 13-acetate and calcium ionophore A23187. Scavenging or inhibition of RNS formation diminished NETs release triggered by these stimuli while scavenging of peroxynitrite inhibited NO-induced NETs formation. Our data suggest that RNS may act as mediators and inducers of NETs release. These processes are PI3K-dependent and ROS-dependent. Since inflammatory reactions are often accompanied by nitrosative stress and NETs formation, our studies shed a new light on possible mechanisms engaged in various immune-mediated conditions.

Neutrophil Extracellular Traps (NETs) in Severe SARS-CoV-2 Lung Disease.

Neutrophil extracellular traps (NETs), built from mitochondrial or nuclear DNA, proteinases, and histones, entrap and eliminate pathogens in the course of bacterial or viral infections. Neutrophils' activation and the formation of NETs have been described as major risk factors for acute lung injury, multi-organ damage, and mortality in COVID-19 disease. NETs-related lung injury involves both epithelial and endothelial cells, as well as the alveolar-capillary barrier. The markers for NETs formation, such as circulating DNA, neutrophil elastase (NE) activity, or myeloperoxidase-DNA complexes, were found in lung specimens of COVID-19 victims, as well as in sera and tracheal aspirates obtained from COVID-19 patients. DNA threads form large conglomerates causing local obstruction of the small bronchi and together with NE are responsible for overproduction of mucin by epithelial cells. Various components of NETs are involved in the pathogenesis of cytokine storm in SARS-CoV-2 pulmonary disease. NETs are responsible for the interplay between inflammation and thrombosis in the affected lungs. The immunothrombosis, stimulated by NETs, has a poor prognostic significance. Better understanding of the role of NETs in the course of COVID-19 can help to develop novel approaches to the therapeutic interventions in this condition.

Extracellular Vesicles in Allergic Rhinitis and Asthma and Laboratory Possibilities for Their Assessment.

Currently, extracellular vesicles (EVs) have been implicated in the etiopathogenesis of many diseases, including lung disorders, with the possibility of diagnostic and therapeutic applications. The analysis of EV in respiratory tract diseases faces many obstacles, including material collection from airways, standardization of isolation techniques, detection methods, the analysis of their content, etc. This review focuses on the role of extracellular vesicles in the pathogenesis of atopic respiratory diseases, especially asthma, with a special focus on their clinical applicability as a diagnostic tool. We also summarize available laboratory techniques that enable the detection of EVs in various biological materials, with particular emphasis on flow cytometry. The opportunities and limitations of detecting EV in bronchoalveolar lavage fluid (BALF) were also described.

Dynamic Changes in the Ability to Release Neutrophil ExtraCellular Traps in the Course of Childhood Acute Leukemias.

Acute leukemias, the most common cancers in children, are characterized by excessive proliferation of malignant progenitor cells. As a consequence of impaired blood cell production, leukemia patients are susceptible to infectious complications-a major cause of non-relapse mortality. Neutrophil extracellular traps (NETs) are involved in various pathologies, from autoimmunity to cancer. Although aberrant NETs formation may be partially responsible for immune defects observed in acute leukemia, still little is known on the NET release in the course of leukemia. Here, we present the first comprehensive evaluation of NETs formation by neutrophils isolated from children with acute leukemia in different stages of the disease and treatment stimulated in vitro with phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore (CI). NETs release was measured using quantitative fluorescent method and visualized microscopically. In this setting, NETs release was significantly impaired in leukemic children both at the diagnosis and during the treatment, and full restoration of neutrophil function was achieved only after successful completion of the leukemia treatment. We suggest that neutrophil function impairment may result from both disease- and treatment-related factors. In this context, deficient innate immune response observed in acute leukemia patients may be present regardless of neutrophil count and contribute to secondary immunodeficiency observed in this population.

Influence of iron- and zinc-chelating agents on neutrophil extracellular trap formation.

Release of neutrophil extracellular traps (NETs) is one of the neutrophils' mechanisms involved in the response to infection. NETs are released from the cell in response to a biological or synthetic stimulus to entrap, immobilize and kill pathogens. Metal ions and metal binding proteins were identified in the structure of NETs, but their role in NET release remains unclear. The aim of this study was to assess how lack of iron and zinc generated by ion sequestration using chelators affects NET release. Neutrophils were isolated from whole blood or buffy coats of healthy blood donors by density gradient centrifugation and incubated with zinc chelators: 20 µM N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), 40 µM diethylenetriaminepentaacetic acid (DTPA) or iron chelators: 400 µM deferoxamine mesylate salt (DFO) and 50 µM iminodiacetic acid (IDA). Next, 100 nM phorbol 12-myristate 13-acetate (PMA) was added to stimulate release of NETs. The amount of released DNA was measured by fluorometry and NETs were visualized by immunofluorescence microscopy. This study demonstrates that iron and zinc chelators are able to modulate NET release. Here we show that preincubation of neutrophils with TPEN and IDA inhibits NET release in cells stimulated with PMA. On the other hand, DFO stimulates NET release. Incubation of cells with DTPA does not affect release of NETs.

Serum neutrophil gelatinase-associated lipocalin for predicting anemia of inflammation in children with urinary tract infection.

Anemia of inflammation (IA), the second most common cause of childhood anemia, results from macrophage iron sequestration and impaired erythropoiesis. Neutrophil gelatinase-associated lipocalin (NGAL) plays an important role in innate microbial immunity through its influence on intracellular iron homeostasis and inhibition of erythropoiesis. The predictive value of NGAL in IA was assessed in 74 children (age 6.30 ±3.64 months) with the first episode of urinary tract infection (UTI). Anemia of inflammation was found in 50% of children, including those with non-febrile UTI, and delayed onset of anemia was observed in 20% of children. There were no differences in NGAL levels between the anemic and non-anemic children, and no correlations between NGAL and hemoglobin (HGB) levels and red blood cell (RBC) count. In multivariate logistic regression analysis elevated C-reactive protein (CRP) was only independently associated with the presence of anemia in children with UTI [OR (95% CI): 1.128 (1.005-1.265), p = 0.040]. In stepwise multiple analysis age independently correlated with RBC (ß = 0.051, p = 0.001), while CRP independently correlated with HGB (ß = -0.037, p = 0.027) and RBC (ß = -0.022, p = 0.014). ROC analysis demonstrated better diagnostic profiles for CRP, procalcitonin (PCT) and fever duration for predicting the risk of IA than NGAL (AUC: 0.690, 0.669, 0.678 vs. 0.638, respectively). Despite the increase in HGB levels after 4-5 weeks from the onset of UTI, HGB values were still significantly lower in the anemic than in non-anemic children. NGAL was not useful for predicting IA in UTI, since its diagnostic value was inferior to conventional inflammatory markers.

Clinical Course and Outcome of Community-Acquired Bacterial Meningitis in Cancer Patients.

The aim of the study was to determine the course and outcome of bacterial meningitis (BM) in patients with cancer. We retrospectively reviewed files of patients with community-acquired BM, hospitalized in a single neuroinfection center between January 2010 and December 2017. There were 209 patients included in the analysis: 28 had cancer (9 women, 19 men; median age 76, IQR 67-80 years) and 181 were cancer-free (76 women, 105 men; median age 52, IQR 33-65 years) and constituted the control group. Cancer patients, compared with controls, were more likely to present with seizures (25% vs. 8%, p = 0.019), scored higher on the Sequential Organ Failure Assessment, and had a higher mortality rate (32% vs. 13%, p = 0.025). Further, cancer patients were less likely (64% vs. 83%, p = 0.033) to present with two or more out of four clinical manifestations of BM (pyrexia, neck stiffness, altered mental status, and headache) and had a lower white blood cell (WBC) count than non-cancer controls. In multiple regression analysis, the presence of bacterial meningitis in cancer patients was independently associated only with older age (p = 0.001) and lower WBC count (p = 0.007), while mortality was associated with lower Glasgow Coma Score (p = 0.003). In conclusion, bacterial meningitis in cancer patients is characterized by atypical symptoms and high mortality, which requires physicians' vigilance and a prompt investigation of cerebrospinal fluid in suspected cases. However, multiple regression analysis suggests that differences in clinical presentation and outcomes of bacterial meningitis between cancer and cancer-free patients may also be attributable to other factors, such as age differences.

Correlation of bronchoalveolar lavage lymphocyte count with the extent of lung fibrosis and with plethysmographic lung volumes in patients with newly recognized hypersensitivity pneumonitis.

INTRODUCTION: Hypersensitivity pneumonitis (HP) is an increasingly recognized interstitial lung disease, presenting with elevated total cell counts and high percentage of lymphocytes in bronchoalveolar lavage fluid (BALF). Despite many publications, there is no consensus in the literature concerning BALF cellular composition in patients with prolonged course of HP. AIM: The aim of the present retrospective study was to investigate the influence of disease duration, smoking habits, and the extent of lung fibrosis on BALF cells' population in patients with newly recognized HP. MATERIAL AND METHODS: In total, 94 patients (49 females, 45 males), mean age 52 (±12) years, with HP recognized according to recently proposed criteria, were enrolled into the present study. Chest CT scans were retrospectively reviewed by two independent radiologists. BALF evaluation was performed as a part of routine diagnostics according to recent recommendations. RESULTS: Percentage of lymphocytes in BALF was significantly lower in patients with lung fibrosis (stage 1 and 2) comparing to those without lung fibrosis (stage 0). Significant correlation was also found between the percentage of BALF lymphocytes and plethysmographic lung volumes, but not with lung transfer capacity for carbon monoxide (TLCO% pred). Smoking did not influence BALF results in our study group. CONCLUSIONS: BALF lymphocytosis correlated with the presence and the extent of lung fibrosis on chest CT as well as with plethysmographic lung volumes but did not with TLCO and smoking habits in newly recognized HP pneumonitis.

Vitamin D and Calcium Homeostasis in Infants with Urolithiasis.

The incidence of urolithiasis in infants is unknown. The aim of this study was to investigate clinical characteristics, nutrition, calcium, phosphate, 25-hydroxyvitamin D (25(OH)D), alkaline phosphate, and parathyroid hormone in infants with urolithiasis. There were 32 infants (23 boys and 9 girls) of the mean age of 6.4 ± 3.7 months (range 2-12 months), with diagnosis of urolithiasis enrolled into the study. Boys were younger than girls (5.3 vs. 9.1 months, respectively; p < 0.05). The infants were receiving prophylactic vitamin D3. Twenty-one of them were fed with milk formula, 9 were breastfed, and 2 were on a mixed diet. The major clinical symptoms consisted of irritability in 19 (59%) and urinary tract infection in 6 (19%) infants. Hypercalcemia and hyperphosphatemia were detected in the serum in 30 (94%) and 19 (60%) infants, respectively. The serum calcium level was higher in boys than girls (10.8 vs. 9.8 mg/dL, respectively; p < 0.05). Four (12.5%) infants had increased activity of alkaline phosphatase. The serum level of 25(OH)D was high in 3 (9%), low in 2 (6%), and normal in 27 (85%) infants. Parathyroid hormone was low in eight (25%) infants. Hypercalciuria and hyperphosphaturia were found in 11 (34%) boys and 8 (25%) girls. Family history of urolithiasis was positive in eight (25%) infants. We conclude that urolithiasis occurs in infancy more often in boys fed with milk formula and in those who received vitamin D supplementation. Hypercalcemia, hyperphosphatemia, and hypercalciuria are the most common changes present in clinical metabolic tests.