Beta-Lactam Allergy Association with Surgical Site Infections in Pediatric Procedures: A Matched Cohort Study.

BACKGROUND: Little is known about surgical site infection (SSI) risk among pediatric patients with reported beta-lactam allergy (BLA). METHODS: We performed a retrospective cohort study at a quaternary children's hospital and compared procedures in patients ages 1-19 years old with and without BLA that required antimicrobial prophylaxis (AMP) during 2010-2017. Procedures were matched 1:1 by patient age, complex chronic conditions, year of surgery, and National Surgical Quality Improvement Program current procedural terminology category. The primary outcome was SSI as defined by National Healthcare Safety Network. The secondary outcome was AMP protocol compliance as per American Society of Health-System Pharmacists. RESULTS: Of the 11 878 procedures identified, 1021 (9%) had a reported BLA. There were 35 (1.8%) SSIs in the matched cohort of 1944 procedures with no significant difference in SSI rates in BLA procedures (1.8%) compared to no-BLA (1.9%) procedures. Tier 3 AMP was chosen more frequently among BLA procedures (P < .01). Unmatched analysis of all procedures showed that 23.7% of BLA procedures received beta-lactam-AMP (vs. 93.7% of procedures without BLA). There were no major differences in SSI on sensitivity analysis of BLA procedures that did not receive beta-lactam AMP (1.4%) compared to no-BLA procedures with beta-lactam AMP (1.6%). CONCLUSIONS: Our retrospective matched analysis of 1944 pediatric procedures found no increase in SSIs in procedures with reported BLA, which differs from studies in adults. We observed that the choice of beta-lactam-AMP was common, even in BLA procedures. More data are needed to delineate an association between non-beta-lactam AMP and SSI in children.

Respiratory Infections in Patients with Primary Immunodeficiency.

Recurrent and life-threatening respiratory infections are nearly universal in patients with primary immunodeficiency diseases (PIDD). Early recognition, aggressive treatment, and prophylaxis with antimicrobials and immunoglobulin replacement have been the mainstays of management and will be reviewed here with an emphasis on respiratory infections. Genetic discoveries have allowed direct translation of research to clinical practice, improving our understanding of clinical patterns of pathogen susceptibilities and guiding prophylaxis. The recent identification of inborn errors in type I interferon signaling as a basis for life-threatening viral infections in otherwise healthy individuals suggests another targetable pathway for treatment and/or prophylaxis. The future of PIDD diagnosis will certainly involve early genetic identification by newborn screening before onset of infections, with early treatment offering the potential of preventing disease complications such as chronic lung changes. Gene editing approaches offer tremendous therapeutic potential, with rapidly emerging delivery systems. Antiviral therapies are desperately needed, and specific cellular therapies show promise in patients requiring hematopoietic stem cell transplantation. The introduction of approved therapies for clinical use in PIDD is limited by the difficulty of studying outcomes in rare patients/conditions with conventional clinical trials.

Frequency and spectrum of disease-causing variants in 1892 patients with suspected genetic HLH disorders.

This article explores the distribution and mutation spectrum of potential disease-causing genetic variants in hemophagocytic lymphohistiocytosis (HLH)-associated genes observed in a large tertiary clinical referral laboratory. Samples from 1892 patients submitted for HLH genetic analysis were studied between September 2013 and June 2018 using a targeted next-generation sequencing panel approach. Patients ranged in age from 1 day to 78 years. Analysis included 15 genes associated with HLH. A potentially causal genetic finding was observed in 227 (12.0%) samples in this cohort. A total of 197 patients (10.4%) had a definite genetic diagnosis. Patients with pathogenic variants in familial HLH genes tended to be diagnosed significantly younger compared with other genes. Pathogenic or likely pathogenic variants in the PRF1 gene were the most frequent. However, mutations in genes associated with degranulation defects (STXBP2, UNC13D, RAB27A, LYST, and STX11) were more common than previously appreciated and collectively represented >50% of cases. X-linked conditions (XIAP, SH2D1A, and MAGT1) accounted for 17.8% of the 197 cases. Pathogenic variants in the SLC7A7 gene were the least encountered. These results describe the largest cohort of genetic variation associated with suspected HLH in North America. Merely 10.4% of patients were identified with a clearly genetic cause by this diagnostic approach; other possible etiologies of HLH should be investigated. These results suggest that careful thought should be given regarding whether patients have a clinical phenotype most consistent with HLH vs other clinical and disease phenotypes. The gene panel identified known pathogenic and novel variants in 10 HLH-associated genes.

Hemophagocytic Lymphohistiocytosis: Clinical Presentations and Diagnosis.

Hemophagocytic lymphohistiocytosis (HLH) is an overwhelming clinical syndrome associated with extreme immune activation. Familial HLH is caused by autosomal-recessive inheritance of gene mutations that cripple lymphocyte cytotoxicity. X-linked lymphoproliferative diseases and mutations in Nod-like receptor caspase activation and recruitment domain containing protein 4 (NLRC4) also feature HLH as a predominant manifestation. In addition, "secondary" HLH may occur in immunocompromized patients or in individuals with previously intact immune responses in the context of strong immunologic triggers such as EBV infection, malignancy, rheumatologic disease, and drug hypersensitivity. Regardless of the etiology, HLH is often fatal unless recognized and treated aggressively. Research over the last 20 years has led to many advances in diagnosis and treatment. Rapid testing strategies designed to quickly screen for immune activation and cytotoxic lymphocyte dysfunction are now clinically available and genetic panels/testing algorithms may accelerate a genetic diagnosis. Immunosuppressive treatment protocols have been refined, and experience is gaining with alternative and salvage approaches. However, these advances improve the outcome of patients only when the diagnosis of HLH is made. Ongoing education is needed to ensure medical providers can appropriately recognize and diagnose HLH. This Grand Rounds Review will summarize the clinical and diagnostic features of HLH and highlight known genetic causes.

Aberrant tRNA processing causes an autoinflammatory syndrome responsive to TNF inhibitors.

OBJECTIVES: To characterise the clinical features, immune manifestations and molecular mechanisms in a recently described autoinflammatory disease caused by mutations in TRNT1, a tRNA processing enzyme, and to explore the use of cytokine inhibitors in suppressing the inflammatory phenotype. METHODS: We studied nine patients with biallelic mutations in TRNT1 and the syndrome of congenital sideroblastic anaemia with immunodeficiency, fevers and developmental delay (SIFD). Genetic studies included whole exome sequencing (WES) and candidate gene screening. Patients' primary cells were used for deep RNA and tRNA sequencing, cytokine profiling, immunophenotyping, immunoblotting and electron microscopy (EM). RESULTS: We identified eight mutations in these nine patients, three of which have not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly interleukin (IL)-6, interferon gamma (IFN-γ) and IFN-induced cytokines were elevated in the serum, whereas tumour necrosis factor (TNF) and IL-1ß were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients' fibroblasts showed significant deficiency of mature cytosolic tRNAs. EM of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for blood transfusions and improved growth. CONCLUSIONS: Mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease consequences.

High Level of Perforin Expression Is Required for Effective Correction of Hemophagocytic Lymphohistiocytosis.

Perforin-1 mutations result in a potentially fatal hemophagocytic lymphohistiocytosis (HLH) with heightened immune activation, hypercytokinemia, pancytopenia, and end-organ damage. At present, hematopoietic stem cell (HSC) transplantation is curative, but limited by donor availability and associated mortality, making gene therapy an attractive alternative approach for HLH. We reported that perforin expression driven by cellular promoters in lentiviral (LV) vectors resulted in significant, albeit partial, correction of the inflammatory features in a murine model of HLH. We hypothesized that the level of perforin expression achieved per cell from ectopic moderate-strength cellular promoters (phosphoglycerate kinase gene/perforin-1 gene) is inadequate and thus engineered an LV vector using a viral promoter (MND; a modified Moloney murine leukemia virus long terminal repeat with myeloproliferative sarcoma virus enhancer) containing microRNA126 target sequences to restrict perforin expression in HSCs. We show here that the MND-LV vector restored perforin expression to normal levels in a perforin-deficient human natural killer cell line and perforin gene-corrected Perforin1-/- transplant recipients, whereas cellular promoters drove only partial correction. On lymphocytic choriomeningitis virus challenge, the clinical scores and survival improved only with the MND-LV vector, but inflammatory markers and cytotoxicity were improved with all LV vectors. Our studies suggest that although moderate levels of expression can result in partial amelioration of the HLH phenotype, high levels of perforin expression per cell are required for complete correction of HLH.

Defects of B-cell terminal differentiation in patients with type-1 Kabuki syndrome.

BACKGROUND: Kabuki syndrome (KS) is a complex multisystem developmental disorder associated with mutation of genes encoding histone-modifying proteins. In addition to craniofacial, intellectual, and cardiac defects, KS is also characterized by humoral immune deficiency and autoimmune disease, yet no detailed molecular characterization of the KS-associated immune phenotype has been reported. OBJECTIVE: We sought to characterize the humoral immune defects found in patients with KS with lysine methyltransferase 2D (KMT2D) mutations. METHODS: We comprehensively characterized B-cell function in a cohort (n = 13) of patients with KS (age, 4 months to 27 years). RESULTS: Three quarters (77%) of the cohort had a detectable heterozygous KMT2D mutation (50% nonsense, 20% splice site, and 30% missense mutations), and 70% of the reported mutations are novel. Among the patients with KMT2D mutations (KMT2D(Mut/+)), hypogammaglobulinemia was detected in all but 1 patient, with IgA deficiency affecting 90% of patients and a deficiency in at least 1 other isoform seen in 40% of patients. Numbers of total memory (CD27(+)) and class-switched memory B cells (IgM(-)) were significantly reduced in patients with KMT2D(Mut/+) mutations compared with numbers in control subjects (P < .001). Patients with KMT2D(Mut/+) mutations also had significantly reduced rates of somatic hypermutation in IgG (P = .003) but not IgA or IgM heavy chain sequences. Impaired terminal differentiation was noted in primary B cells from patients with KMT2D(Mut/+) mutations. Autoimmune pathology was observed in patients with missense mutations affecting the SET domain and its adjacent domains. CONCLUSIONS: In patients with KS, autosomal dominant KMT2D mutations are associated with dysregulation of terminal B-cell differentiation, leading to humoral immune deficiency and, in some cases, autoimmunity. All patients with KS should undergo serial clinical immune evaluations.

Live cell evaluation of granzyme delivery and death receptor signaling in tumor cells targeted by human natural killer cells.

Growing interest in natural killer (NK) cell-based therapy for treating human cancer has made it imperative to develop new tools to measure early events in cell death. We recently demonstrated that protease-cleavable luciferase biosensors detect granzyme B and pro-apoptotic caspase activation within minutes of target cell recognition by murine cytotoxic lymphocytes. Here we report successful adaptation of the biosensor technology to assess perforin-dependent and -independent induction of death pathways in tumor cells recognized by human NK cell lines and primary cells. Cell-cell signaling via both Fc receptors and NK-activating receptors led to measurable luciferase signal within 15 minutes. In addition to the previously described aspartase-cleavable biosensors, we report development of granzyme A and granzyme K biosensors, for which no other functional reporters are available. The strength of signaling for granzyme biosensors was dependent on perforin expression in IL-2-activated NK effectors. Perforin-independent induction of apoptotic caspases was mediated by death receptor ligation and was detectable after 45 minutes of conjugation. Evidence of both FasL and TRAIL-mediated signaling was seen after engagement of Jurkat cells by perforin-deficient human cytotoxic lymphocytes. Although K562 cells have been reported to be insensitive to TRAIL, robust activation of pro-apoptotic caspases by NK cell-derived TRAIL was detectable in K562 cells. These studies highlight the sensitivity of protease-cleaved luciferase biosensors to measure previously undetectable events in live cells in real time. Further development of caspase and granzyme biosensors will allow interrogation of additional features of granzyme activity in live cells including localization, timing, and specificity.

Caustic ingestions mimicking anaphylaxis: case studies and literature review.

Anaphylaxis presents in children with rapid involvement of typically 2 or more organ systems including cutaneous, gastrointestinal, and respiratory. Caustic ingestions (CI) may also present with acute involvement of cutaneous, gastrointestinal, and respiratory systems. We present 2 cases of "missed diagnosis" that illustrate how CI presenting with respiratory symptoms can be mistaken for anaphylaxis owing to these similarities. Both of these patients had delay in appropriate care for CI as a result. These cases demonstrate the importance of considering CI in children who have gastrointestinal symptoms, respiratory distress, and oropharyngeal edema.

Perforin gene transfer into hematopoietic stem cells improves immune dysregulation in murine models of perforin deficiency.

Defects in perforin lead to the failure of T and NK cell cytotoxicity, hypercytokinemia, and the immune dysregulatory condition known as familial hemophagocytic lymphohistiocytosis (FHL). The only curative treatment is allogeneic hematopoietic stem cell transplantation which carries substantial risks. We used lentiviral vectors (LV) expressing the human perforin gene, under the transcriptional control of the ubiquitous phosphoglycerate kinase promoter or a lineage-specific perforin promoter, to correct the defect in different murine models. Following LV-mediated gene transfer into progenitor cells from perforin-deficient mice, we observed perforin expression in mature T and NK cells, and there was no evidence of progenitor cell toxicity when transplanted into irradiated recipients. The resulting perforin-reconstituted NK cells showed partial recovery of cytotoxicity, and we observed full recovery of cytotoxicity in polyclonal CD8(+) T cells. Furthermore, reconstituted T cells with defined antigen specificity displayed normal cytotoxic function against peptide-loaded targets. Reconstituted CD8(+) lymphoblasts had reduced interferon-γ secretion following stimulation in vitro, suggesting restoration of normal immune regulation. Finally, upon viral challenge, mice with >30% engraftment of gene-modified cells exhibited reduction of cytokine hypersecretion and cytopenias. This study demonstrates the potential of hematopoietic stem cell gene therapy as a curative treatment for perforin-deficient FHL.

Real-time detection of CTL function reveals distinct patterns of caspase activation mediated by Fas versus granzyme B.

Activation of caspase-mediated apoptosis is reported to be a hallmark of both granzyme B- and Fas-mediated pathways of killing by CTLs; however, the kinetics of caspase activation remain undefined owing to an inability to monitor target cell-specific apoptosis in real time. We have overcome this limitation by developing a novel biosensor assay that detects continuous, protease-specific activity in target cells. Biosensors were engineered from a circularly permuted luciferase, linked internally by either caspase 3/7 or granzyme B/caspase 8 cleavage sites, thus allowing activation upon proteolytic cleavage by the respective proteases. Coincubation of murine CTLs with target cells expressing either type of biosensor led to a robust luminescent signal within minutes of cell contact. The signal was modulated by the strength of TCR signaling, the ratio of CTL/target cells, and the type of biosensor used. Additionally, the luciferase signal at 30 min correlated with target cell death, as measured by a (51)Cr-release assay. The rate of caspase 3/7 biosensor activation was unexpectedly rapid following granzyme B- compared with Fas-mediated signal induction in murine CTLs; the latter appeared gradually after a 90-min delay in perforin- or granzyme B-deficient CTLs. Remarkably, the Fas-dependent, caspase 3/7 biosensor signal induced by perforin-deficient human CTLs was also detectable after a 90-min delay when measured by redirected killing. Thus, we have used a novel, real-time assay to demonstrate the distinct pattern of caspase activation induced by granzyme B versus Fas in human and murine CTLs.

Hemophagocytic lymphohistiocytosis: updates and evolving concepts.

PURPOSE OF REVIEW: Hemophagocytic lymphohistiocytosis (HLH) is an immune dysregulatory syndrome that is associated with underlying defects of perforin-dependent cytotoxic function. This review seeks to update readers on new scientific insights and evolving clinical concepts related to this rare but fatal disorder. RECENT FINDINGS: Clinically, HLH is defined by severe inflammation and potentially fatal damage to a variety of organ systems including the bone marrow, liver, or brain. Recent preclinical studies have increasingly defined HLH as a syndrome of abnormal and excessive T-cell activation, which leads to toxic activation of macrophages and other innate immune cells. Although macrophages have long been suspected to be important for disease development, recent studies have for the first time demonstrated their central role in the development of inflammation-associated cytopenias. In addition to defining new therapeutic targets, these scientific insights suggest significant overlap between HLH and severe inflammation in a variety of clinical contexts. Recent clinical observations are also changing how HLH is conceptualized. Increased recognition of HLH in older children and adults, sometimes in association with classic disease-associated mutations, is challenging the traditional view of HLH as either a distinctly familial or a sporadic disorder. SUMMARY: Recent scientific and clinical insights are expanding understanding and recognition of HLH, driving an evolution in how it is defined, and suggesting future directions for improving therapy of this disorder.

Hypomorphic mutations in PRF1, MUNC13-4, and STXBP2 are associated with adult-onset familial HLH.

Familial hemophagocytic lymphohistiocytosis (HLH) is a rare primary immunodeficiency disorder characterized by defects in cell-mediated cytotoxicity that results in fever, hepatosplenomegaly, and cytopenias. Familial HLH is well recognized in children but rarely diagnosed in adults. We conducted a retrospective review of genetic and immunologic test results in patients who developed HLH in adulthood. Included in our study were 1531 patients with a clinical diagnosis of HLH; 175 patients were 18 years or older. Missense and splice-site sequence variants in PRF1, MUNC13-4, and STXBP2 were found in 25 (14%) of the adult patients. The A91V-PRF1 genotype was found in 12 of these patients (48%). The preponderance of hypomorphic mutations in familial HLH-causing genes correlates with the later-onset clinical symptoms and the more indolent course in adult patients. We conclude that late-onset familial HLH occurs more commonly than was suspected previously.

Perforin is a critical physiologic regulator of T-cell activation.

Individuals with impaired perforin-dependent cytotoxic function (Ctx(-)) develop a fatal inflammatory disorder called hemophagocytic lymphohistiocytosis (HLH). It has been hypothesized that immune hyperactivation during HLH is caused by heightened infection, defective apoptosis/responsiveness of Ctx(-) lymphocytes, or enhanced antigen presentation. Whereas clinical and experimental data suggest that increased T-cell activation drives HLH, potential abnormalities of T-cell activation have not been well characterized in Ctx(-) hosts. To define such abnormalities and to test these hypotheses, we assessed in vivo T-cell activation kinetics and viral loads after lymphocytic choriomeningitis virus (LCMV) infection of Ctx(-) mice. We found that increased T-cell activation occurred early during infection of Ctx(-) mice, while they had viral burdens that were identical to those of WT animals, demonstrating that T-cell hyperactivation was independent of viral load. Furthermore, cell transfer and signaling studies indicated that increased antigenic stimulation, not a cell-intrinsic defect of responsiveness, underlay heightened T-cell activation in vivo. Finally, direct measurement of viral antigen presentation demonstrated an increase in Ctx(-) mice that was proportional to abnormal T-cell activation. We conclude that perforin-dependent cytotoxicity has an immunoregulatory role that is distinguishable from its pathogen clearance function and limits T-cell activation in the physiologic context by suppressing antigen presentation.

Gene expression profiling of peripheral blood mononuclear cells from children with active hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare, genetically heterogeneous autosomal recessive immune disorder that results when the critical regulatory pathways that mediate immune defense mechanisms and the natural termination of immune/inflammatory responses are disrupted or overwhelmed. To advance the understanding of FHL, we performed gene expression profiling of peripheral blood mononuclear cells from 11 children with untreated FHL. Total RNA was isolated and gene expression levels were determined using microarray analysis. Comparisons between patients with FHL and normal pediatric controls (n = 30) identified 915 down-regulated and 550 up-regulated genes with more than or equal to 2.5-fold difference in expression (P ≤ .05). The expression of genes associated with natural killer cell functions, innate and adaptive immune responses, proapoptotic proteins, and B- and T-cell differentiation were down-regulated in patients with FHL. Genes associated with the canonical pathways of interleukin-6 (IL-6), IL-10 IL-1, IL-8, TREM1, LXR/RXR activation, and PPAR signaling and genes encoding of antiapoptotic proteins were overexpressed in patients with FHL. This first study of genome-wide expression profiling in children with FHL demonstrates the complexity of gene expression patterns, which underlie the immunobiology of FHL.

Hypohidrotic ectodermal dysplasia and immunodeficiency with coincident NEMO and EDA mutations.

Ectodermal dysplasias (ED) are uncommon genetic disorders resulting in abnormalities in ectodermally derived structures. Many ED-associated genes have been described, of which ectodysplasin-A (EDA) is one of the more common. The NF-κB essential modulator (NEMO encoded by the IKBKG gene) is unique in that mutations result in severe humoral and cellular immunologic defects in addition to ED. We describe three unrelated kindreds with defects in both EDA and IKBKG resulting from X-chromosome crossover. This demonstrates the importance of thorough immunologic consideration of patients with ED even when an EDA etiology is confirmed, and raises the possibility of a specific phenotype arising from coincident mutations in EDA and IKBKG.

Patients with X-linked lymphoproliferative disease due to BIRC4 mutation have normal invariant natural killer T-cell populations.

Human invariant natural killer T cells (iNKT cells) are a unique population of T cells that express an invariantly rearranged T-cell receptor (TCR) composed of TCRValpha24 and TCRVbeta11 chains which recognize glycosphingolipid antigens presented by the CD1d molecule. iNKT cells are absent in patients with X-linked lymphoproliferative disease (XLP) due to SH2D1A mutation, and are reported to be decreased in patients with XLP due to BIRC4 mutations. However, mice deficient in the BIRC4 gene product, X-linked Inhibitor of Apoptosis (XIAP), have normal iNKT cell populations. Because of this, we studied iNKT cell populations in 6 patients with XLP due to BIRC4 mutations, with comparison to 103 pediatric and adult normal control samples. We found that iNKT cells constitute 0.008%-1.176% of normal peripheral blood T cells, and that iNKT cell populations were normal or increased in patients with BIRC4 mutations. We conclude that XLP due to BIRC4 mutation is not associated with decreased populations of iNKT cells, and that XIAP is likely not a requirement for iNKT cell development.

A rapid flow cytometric screening test for X-linked lymphoproliferative disease due to XIAP deficiency.

BACKGROUND: Deficiency of X-linked inhibitor of apoptosis (XIAP), caused by BIRC4 gene mutations, is the second known cause of X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency that often presents with life-threatening hemophagocytic lymphohistiocytosis (HLH). Rapid diagnosis of the known genetic causes of HLH, including XIAP deficiency, facilitates the initiation of life-saving treatment and preparation for allogeneic hematopoietic cell transplantation (HCT). Until now, a rapid screening test for XIAP deficiency has not been available. METHODS: To develop a flow cytometric screening test for XIAP deficiency, we first used lymphoblastic cell lines generated from controls and patients with BIRC4 mutations to identify two commercially available antibodies specific for native intracellular XIAP. Next, we used these antibodies to study control whole blood leukocyte XIAP expression. We then studied XIAP expression in leukocytes from patients with XLP due to BIRC4 mutations, maternal carriers, and patients following HCT. RESULTS: XIAP was expressed by the majority of all whole blood nucleated cells in normal controls. In contrast, XIAP was absent or decreased in all lymphocyte subsets, monocytes and granulocytes from four unrelated patients with XLP due to BIRC4 mutations. Bimodal distribution of XIAP expression was evident in two maternal carriers, with significant skewing toward cells expressing normal XIAP. Bimodal distribution was also observed in a patient following HCT. CONCLUSIONS: Flow cytometric analysis of intracellular XIAP provides a rapid screening test for XLP due to XIAP deficiency. It also allows carrier detection and can be used to monitor donor versus recipient reconstitution following HCT.

Functional assessment of perforin C2 domain mutations illustrates the critical role for calcium-dependent lipid binding in perforin cytotoxic function.

Perforin-mediated lymphocyte cytotoxicity is critical for pathogen elimination and immune homeostasis. Perforin disruption of target cell membranes is hypothesized to require binding of a calcium-dependent, lipid-inserting, C2 domain. In a family affected by hemophagocytic lymphohistiocytosis, a severe inflammatory disorder caused by perforin deficiency, we identified 2 amino acid substitutions in the perforin C2 domain: T435M, a previously identified mutant with disputed pathogenicity, and Y438C, a novel substitution. Using biophysical modeling, we predicted that the T435M substitution, but not Y438C, would interfere with calcium binding and thus cytotoxic function. The capacity for cytotoxic function was tested after expression of the variant perforins in rat basophilic leukemia cells and murine cytotoxic T lymphocytes. As predicted, cells transduced with perforin-T435M lacked cytotoxicity, but those expressing perforin-Y438C displayed intact cytotoxic function. Using novel antibody-capture and liposome-binding assays, we found that both mutant perforins were secreted; however, only nonmutated and Y438C-substituted perforins were capable of calcium-dependent lipid binding. In addition, we found that perforin-Y438C was capable of mediating cytotoxicity without apparent proteolytic maturation. This study clearly demonstrates the pathogenicity of the T435M mutation and illustrates, for the first time, the critical role of the human perforin C2 domain for calcium-dependent, cytotoxic function.

A kindred of children with interstitial lung disease.

BACKGROUND: Childhood interstitial lung disease (ILD) is a spectrum of diseases including many different rare lung conditions. We present a family with an unusual presentation of ILD in association with rheumatologic and immunologic abnormalities. METHODS: Eight children with a common father were evaluated for evidence of lung disease in association with rheumatologic findings. All underwent routine history and physical examination, hematologic evaluation, and chest radiography and/or CT scan of the chest. Seven children underwent a more extensive immunologic evaluation. Those who were able underwent pulmonary function testing, and four children underwent lung biopsy. RESULTS: Six of eight children with a common father were found to have radiographic findings consistent with ILD. These children also had evidence of autoimmune disease with joint symptoms, alopecia, rheumatoid factor production, and hypergammaglobulinemia. Open-lung biopsy in four children revealed a spectrum of pulmonary lymphoid proliferations ranging from reactive lymphoid hyperplasia to lymphoid interstitial pneumonia. CONCLUSION: The findings of ILD and autoimmunity in a kindred of children suggest a novel genetic disorder of autosomal dominant pattern and variable penetrance. Although the precise pathogenesis remains unclear, these cases provide valuable insight into childhood ILD.