Loss of B Cells in Patients with Heterozygous Mutations in IKAROS.

BACKGROUND: Common variable immunodeficiency (CVID) is characterized by late-onset hypogammaglobulinemia in the absence of predisposing factors. The genetic cause is unknown in the majority of cases, and less than 10% of patients have a family history of the disease. Most patients have normal numbers of B cells but lack plasma cells. METHODS: We used whole-exome sequencing and array-based comparative genomic hybridization to evaluate a subset of patients with CVID and low B-cell numbers. Mutant proteins were analyzed for DNA binding with the use of an electrophoretic mobility-shift assay (EMSA) and confocal microscopy. Flow cytometry was used to analyze peripheral-blood lymphocytes and bone marrow aspirates. RESULTS: Six different heterozygous mutations in IKZF1, the gene encoding the transcription factor IKAROS, were identified in 29 persons from six families. In two families, the mutation was a de novo event in the proband. All the mutations, four amino acid substitutions, an intragenic deletion, and a 4.7-Mb multigene deletion involved the DNA-binding domain of IKAROS. The proteins bearing missense mutations failed to bind target DNA sequences on EMSA and confocal microscopy; however, they did not inhibit the binding of wild-type IKAROS. Studies in family members showed progressive loss of B cells and serum immunoglobulins. Bone marrow aspirates in two patients had markedly decreased early B-cell precursors, but plasma cells were present. Acute lymphoblastic leukemia developed in 2 of the 29 patients. CONCLUSIONS: Heterozygous mutations in the transcription factor IKAROS caused an autosomal dominant form of CVID that is associated with a striking decrease in B-cell numbers. (Funded by the National Institutes of Health and others.).

Activated STING in a vascular and pulmonary syndrome.

BACKGROUND: The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation. METHODS: We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-ß, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls. RESULTS: We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mononuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibroblasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients' lymphocytes was reduced by JAK inhibitors. CONCLUSIONS: STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disease caused by gain-of-function mutations in TMEM173. (Funded by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases; ClinicalTrials.gov number, NCT00059748.).

Mutations in PIK3CD can cause hyper IgM syndrome (HIGM) associated with increased cancer susceptibility.

Autosomal dominant gain of function mutations in the gene encoding PI3K p110δ were recently associated with a novel combined immune deficiency characterized by recurrent sinopulmonary infections, CD4 lymphopenia, reduced class-switched memory B cells, lymphadenopathy, CMV and/or EBV viremia and EBV-related lymphoma. A subset of affected patients also had elevated serum IgM. Here we describe three patients in two families who were diagnosed with HIGM at a young age and were recently found to carry heterozygous mutations in PIK3CD. These patients had an abnormal circulating B cell distribution featuring a preponderance of early transitional (T1) B cells and plasmablasts. When stimulated in vitro, PIK3CD mutated B cells were able to secrete class-switched immunoglobulins. This finding implies that the patients' elevated serum IgM levels were unlikely a product of an intrinsic B cell functional inability to class switch. All three patients developed malignant lymphoproliferative syndromes that were not associated with EBV. Thus, we identified a novel subset of patients with PIK3CD mutations associated with HIGM, despite indications of preserved in vitro B cell class switch recombination, as well as susceptibility to non-EBV-associated malignancies.

Depletion of new neurons by image guided irradiation.

Ionizing radiation continues to be a relevant tool in both imaging and the treatment of cancer. Experimental uses of focal irradiation have recently been expanded to studies of new neurons in the adult brain. Such studies have shown cognitive deficits following radiation treatment and raised caution as to possible unintentional effects that may occur in humans. Conflicting outcomes of the effects of irradiation on adult neurogenesis suggest that the effects are either transient or permanent. In this study, we used an irradiation apparatus employed in the treatment of human tumors to assess radiation effects on rat neurogenesis. For subjects we used adult male rats (Sprague-Dawley) under anesthesia. The irradiation beam was directed at the hippocampus, a center for learning and memory, and the site of neurogenic activity in adult brain. The irradiation was applied at a dose-rate 0.6 Gy/min for total single-fraction, doses ranging from 0.5 to 10.0 Gy. The animals were returned to home cages and recovered with no sign of any side effects. The neurogenesis was measured either 1 week or 6 weeks after the irradiation. At 1 week, the number of neuronal progenitors was reduced in a dose-dependent manner with the 50% reduction at 0.78 Gy. The dose-response curve was well fitted by a double exponential suggesting two processes. Examination of the tissue with quantitative immunohistochemistry revealed a dominant low-dose effect on neuronal progenitors resulting in 80% suppression of neurogenesis. This effect was partially reversible, possibly due to compensatory proliferation of the remaining precursors. At higher doses (>5 Gy) there was additional, nearly complete block of neurogenesis without compensatory proliferation. We conclude that notwithstanding the usefulness of irradiation for experimental purposes, the exposure of human subjects to doses often used in radiotherapy treatment could be damaging and cause cognitive impairments.

Regulation of invasive behavior by vascular endothelial growth factor is HEF1-dependent.

We previously reported a vascular endothelial growth factor (VEGF) autocrine loop in head and neck squamous cell carcinoma (HNSCC) cell lines, supporting a role for VEGF in HNSCC tumorigenesis. Using a phosphotyrosine proteomics approach, we screened the HNSCC cell line, squamous cell carcinoma-9 for effectors of VEGFR2 signaling. A cluster of proteins involved in cell migration and invasion, including the p130Cas paralog, human enhancer of filamentation 1 (HEF1/Cas-L/Nedd9) was identified. HEF1 silencing and overexpression studies revealed a role for VEGF in regulating cell migration, invasion and matrix metalloproteinase (MMP) expression in a HEF1-dependent manner. Moreover, cells plated on extracellular matrix-coated coverslips showed enhanced invadopodia formation in response to VEGF that was HEF1-dependent. Immunolocalization revealed that HEF1 colocalized to invadopodia with MT1-MMP. Analysis of HNSCC tissue microarrays for HEF1 immunoreactivity revealed a 6.5-fold increase in the odds of having a metastasis with a high HEF1 score compared with a low HEF1 score. These findings suggest that HEF1 may be prognostic for advanced stage HNSCC. They also show for the first time that HEF1 is required for VEGF-mediated HNSCC cell migration and invasion, consistent with HEF1's recent identification as a metastatic regulator. These results support a strategy targeting VEGF:VEGFR2 in HNSCC therapeutics.

A 475 years-old founder effect involving IL12RB1: a highly prevalent mutation conferring Mendelian Susceptibility to Mycobacterial Diseases in European descendants.

Mutations in IFNGR1, IFNGR2, IL12RB1, IL12B, STAT1 and NEMO result in a common clinical phenotype known as Mendelian Susceptibility to Mycobacterial Diseases (MSMD). Interleukin-12 receptor beta1 (IL-12Rbeta1) deficiency is the most common genetic etiology for MSMD. Known mutations affecting IL12RB1 are recessively inherited and are associated with null response to both IL-12 and IL-23. Mutation IL12RB1 1623_1624delinsTT was originally described in 5 families from European origin (2 from Germany; 1 from Cyprus, France and Belgium). Interestingly, this same mutation was found in an unexpectedly high prevalence among IL-12Rbeta1 deficient patients in Argentina: 5-out-of-6 individuals born to unrelated families carried this particular change. To determine whether mutation 1623_1624delinsTT represents a DNA mutational hotspot or a founder effect, 34 polymorphic markers internal or proximal to IL12RB1 were studied in the Argentinean and the Belgian patients. A common haplotype spanning 1.45-3.51Mb was shared by all chromosomes carrying mutation 1623_1624delinsTT, and was not detected on 100 control chromosomes. Applying a modified likelihood-based method the age of the most recent common ancestor carrying mutation 1623_1624delinsTT was estimated in 475 years (95% CI, 175-1275), which is the time when the Spaniards initiated the colonization of the Americas. Mutation 1623_1624delinsTT represents the first founder effect described on IL-12Rbeta1, the most frequently affected gene in MSMD, and affecting patients with European ancestors. The reason(s) behind the persistency of this mutation across multiple generations, its relative high prevalence, and any potential selective advantage are yet to be established.

Clinical and histopathological features and a unique spectrum of organisms significantly associated with chronic granulomatous disease osteomyelitis during childhood.

Herein, we describe a combination of clinical, microbiologic, and histopathologic findings significantly associated with osteomyelitis in chronic granulomatous disease. When present, these features should raise the suspicion of underlying chronic granulomatous disease. In patients with these findings, anti-infective prophylactic measures aiming to cover highly prevalent microorganisms, as well as aggressive therapeutic measures, should be strongly encouraged.

Inflammatory manifestations in chronic granulomatous disease (CGD).

Chronic granulomatous disease (CGD) is a genetically heterogeneous disease characterized by recurrent life-threatening infections with bacteria and fungi as well as dysregulated inflammatory mechanisms. CGD is caused by defects in the NADPH oxidase, the enzyme complex responsible for generation of superoxide and other reactive oxygen species (ROS) in phagocytic cells. In this review we will focus our attention on those particular inflammatory manifestations associated with CGD, their frequencies and the underlying immunologic mechanisms favoring it occurrence.

A prospective randomized clinical trial of patellar resurfacing and nonresurfacing in bilateral TKA.

UNLABELLED: We conducted a randomized clinical trial to determine long-term outcome differences of patella resurfacing versus nonresurfacing in patients undergoing bilateral total knee arthroplasty. We questioned whether there were differences with respect to the operative procedure, anterior knee pain, Knee Society scores, patellofemoral-related revision rates, patient satisfaction and preference, and patellofemoral functional activities. Thirty-two patients (64 knees) underwent primary bilateral single-stage total knee arthroplasty for osteoarthritis. All patients received the same cruciate-retaining total knee arthroplasty. Patients were randomized to resurfacing or nonresurfacing of the patella for the first total knee arthroplasty, and the second knee received the opposite treatment. All living patients were followed to a minimum of 10 years. We found no differences with regard to range of motion, Knee Society Clinical Rating Score, satisfaction, revision rates, or anterior knee pain. Thirty-seven percent of patients preferred the resurfaced knee, 22% the nonresurfaced knee, and 41% had no preference. Two patients (7.4%) in the nonresurfaced group and one patient (3.5%) in the resurfaced group underwent revision for a patellofemoral-related complication. Equivalent clinical results for resurfaced and nonresurfaced patellae in total knee arthroplasty were demonstrated in this 10-year randomized clinical trial. LEVEL OF EVIDENCE: Level I, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.

Efficacy and tolerability of an argentine intravenous immunoglobulin in pediatric patients with primary immunodeficiency diseases.

Inmunoglobulina G Endovenosa UNC is a 5% liquid Argentine intravenous immunoglobulin obtained from South American donors. This prospective trial was designed to evaluate if the product meets the minimal efficacy requirement of the US Food and Drug Administration of <1 serious infection/subject/year as well as its safety in pediatric patients with Primary Immunodeficiency Diseases. Thirty patients under the age of 18, with well-defined Primary Immunodeficiency Diseases received Inmunoglobulina G Endovenosa UNC (330-700 mg/kg every 3-4 weeks) for 6 months. Vital signs, laboratory abnormalities, adverse events and viral tests were assessed to evaluate safety. Two serious infections occurred (pneumonia and bacteriemia). The estimated infection rate was 0.114 serious infection/subject/year (95% CI, 0.003-0.2277). Minor adverse events occurred in 5.5% of infusions; fever and headache were the most common. Neither severe adverse events, nor abnormal laboratory values were observed. All viral assessments were negative. Inmunoglobulina G Endovenosa UNC meets the minimal efficacy requirement of the US Food and Drug Administration for pediatric Primary Immunodeficiency Diseases patients and showed efficacy and safety data comparable with other data published.

Penicillium piceum infection: diagnosis and successful treatment in chronic granulomatous disease.

Infections due to Penicillium species other than P.marneffei are rare. We identified a boy with X-linked chronic granulomatous disease (X-CGD) with a pulmonary nodule and adjacent rib osteomyelitis caused by Penicillium piceum. The only sign of infection was an elevated sedimentation rate. P. piceum was isolated by fine needle aspirate and from excised infected tissues. Surgical removal and one year of voriconazole treatment were very well tolerated and led to complete recovery. Microbiological, microscopic and molecular studies support the fungal diagnosis. P. piceum should be considered as a relevant pathogen in immunocompromised patients.

Thirteen years of culture-positive M. bovis-BCG infection in an IL-12Rbeta1 deficient patient: treatment and outcome.

Patients with mutations in the IFNgamma/IL-12 pathway show an exquisite susceptibility to mycobacterial diseases. An IL-12Rbeta1 deficient patient with impaired intestinal absorption suffered from a 13 year culture-positive Mycobacterium bovis-BCG infection with acquired multidrug resistance. A combined parenteral and enteral anti-mycobacterial treatment, including recombinant IFNgamma, helped to clear his infection.

Mistletoe in cancer - a systematic review on controlled clinical trials.

BACKGROUND: Mistletoe preparations are among the most widely used unconventional cancer therapies in Central Europe. Their clinical effectiveness, however, is controversial. OBJECTIVE: To investigate whether prospective controlled clinical trials provide evidence for efficacy of mistletoe therapy in cancer. DESIGN: Systematic review. MATERIAL AND METHODS: Search of 11 electronic databases, reference lists and expert consultations. Criteria based analysis was performed to assess methodological quality of the studies. RESULTS: Twenty-three studies were identified: 16 randomized, 2 quasi-randomized and 5 non-randomized. Cancer sites included breast, lung, stomach, colon, rectum, head and neck, kidney, bladder, melanoma, glioma, and genital. Among these studies, statistically significant positive outcomes were reported for survival (n = 8), tumor remission (n = 1), overall quality of life (QOL) (n = 3), and QOL in relation to side effects during cytoreductive therapy (n = 3). Further, positive trends were reported for survival (n = 8), disease-free-survival (n = 1), and tumor remission (n = 2). Several studies reported no effect on survival (n = 4), disease-free-survival (n = 1), recurrence (n = 2), remission (n = 3), and QOL (n = 1). One study showed a negative trend for disease-free-survival. However, methodological quality of the studies was sometimes far below the standard that is today regarded as optimal or necessary. In view of substantial heterogeneity of the studies and potential positive and negative biases, we considered effect size estimation by quantitative synthesis to be unreliable and decided on a non-quantitative synthesis and discussion. Mistletoe therapy was well tolerated, and no major side effects were noted. CONCLUSIONS: Among 23 identified studies evaluated for clinically relevant outcome measures, 12 studies showed one or more statistically significant, positive results, another 7 studies showed at least one positive trend, 3 showed no effect and 1 had a negative trend. All studies, however, suffered from methodological shortcomings to some degree, and many of the studies are not conclusive. As several reasonably well conducted studies indicate beneficial effects, further properly designed trials should be encouraged. Future controlled studies should take into account the methodological limitations and potential biases of these past mistletoe trials.

561del4 defines a novel small deletion hotspot in the interferon-gamma receptor 1 chain.

Patients with a dominant small deletion (818del4, hotspot) in the interferon-gamma receptor 1 (IFNGR1) gene (6q23-q24) and increased susceptibility to mycobacterial infections have been recently reported. We describe a female patient homozygous for a 4-bp deletion in exon 5 of IFNGR1 (561del4) who developed postvaccinal disseminated Bacille Calmette-Guerin infection. She was born to unrelated Argentinean parents, each of whom was heterozygous for this mutation. 561del4 has been previously described as a maternally inherited mutation in a compound heterozygous German patient. By single nucleotide polymorphism analysis of the areas surrounding the deletion, we showed the independent inheritance of 561del4 in three heterozygous carriers. Polypurine runs and "direct repeats," previously shown to be associated with areas of recurrent small deletions, were found in the flanking region of 561del4. The independent inheritance of three identical mutational events defines 561del4 as a new hotspot in the IFNGR1 gene.

Juvenile rheumatoid arthritis-like polyarthritis in Nijmegen breakage syndrome.

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disease (8q21) from the family of the genetically determined chromosomal instability syndromes. The disorder is characterized by microcephaly, growth retardation, immunodeficiency, and high incidence of cancer. Several noninflammatory anomalies of the musculoskeletal system have been described in patients with this syndrome. We describe an Argentinian girl with all the clinical, immunological, and cytogenic characteristics described for NBS plus a juvenile rheumatoid arthritis-like syndrome. To our knowledge this is the first report of a patient with the NBS who presented with a symmetric chronic polyarthritis resembling JRA.

Mindfulness-based stress reduction and health-related quality of life in a heterogeneous patient population.

This study examined the effects of mindfulness-based stress reduction (MBSR) on health-related quality of life and physical and psychological symptomatology in a heterogeneous patient population. Patients (n=136) participated in an 8-week MBSR program and were required to practice 20 min of meditation daily. Pre- and post-intervention data were collected by using the Short-Form Health Survey (SF-36), Medical Symptom Checklist (MSCL) and Symptom Checklist-90 Revised (SCL-90-R). Health-related quality of life was enhanced as demonstrated by improvement on all indices of the SF-36, including vitality, bodily pain, role limitations caused by physical health, and social functioning (all P<.01). Alleviation of physical symptoms was revealed by a 28% reduction on the MSCL (P<.0001). Decreased psychological distress was indicated on the SCL-90-R by a 38% reduction on the Global Severity Index, a 44% reduction on the anxiety subscale, and a 34% reduction on the depression subscale (all P<.0001). One-year follow-up revealed maintenance of initial improvements on several outcome parameters. We conclude that a group mindfulness meditation training program can enhance functional status and well-being and reduce physical symptoms and psychological distress in a heterogeneous patient population and that the intervention may have long-term beneficial effects.

Impairment of mycobacterial but not viral immunity by a germline human STAT1 mutation.

Interferons (IFN) alpha/beta and gamma induce the formation of two transcriptional activators: gamma-activating factor (GAF) and interferon-stimulated gamma factor 3 (ISGF3). We report a natural heterozygous germline STAT1 mutation associated with susceptibility to mycobacterial but not viral disease. This mutation causes a loss of GAF and ISGF3 activation but is dominant for one cellular phenotype and recessive for the other. It impairs the nuclear accumulation of GAF but not of ISGF3 in heterozygous cells stimulated by IFNs. Thus, the antimycobacterial, but not the antiviral, effects of human IFNs are principally mediated by GAF.

Bitter taste transduced by PLC-beta(2)-dependent rise in IP(3) and alpha-gustducin-dependent fall in cyclic nucleotides.

Current evidence points to the existence of multiple processes for bitter taste transduction. Previous work demonstrated involvement of the polyphosphoinositide system and an alpha-gustducin (Galpha(gust))-mediated stimulation of phosphodiesterase in bitter taste transduction. Additionally, a taste-enriched G protein gamma-subunit, Ggamma(13), colocalizes with Galpha(gust) and mediates the denatonium-stimulated production of inositol 1,4,5-trisphosphate (IP(3)). Using quench-flow techniques, we show here that the bitter stimuli, denatonium and strychnine, induce rapid (50-100 ms) and transient reductions in cAMP and cGMP and increases in IP(3) in murine taste tissue. This decrease of cyclic nucleotides is inhibited by Galpha(gust) antibodies, whereas the increase in IP(3) is not affected by antibodies to Galpha(gust). IP(3) production is inhibited by antibodies specific to phospholipase C-beta(2) (PLC-beta(2)), a PLC isoform known to be activated by Gbetagamma-subunits. Antibodies to PLC-beta(3) or to PLC-beta(4) were without effect. These data suggest a transduction mechanism for bitter taste involving the rapid and transient metabolism of dual second messenger systems, both mediated through a taste cell G protein, likely composed of Galpha(gust)/beta/gamma(13), with both systems being simultaneously activated in the same bitter-sensitive taste receptor cell.

Mycobacterial diseases in primary immunodeficiencies.

Primary immunodeficiency diseases comprise over 100 conditions, each associated with a variety of viral, bacterial, fungal and protozoan infections. M. tuberculosis and less virulent mycobacteria, such as bacille Calmette-Guérin vaccines and environmental non-tuberculous mycobacteria, may cause severe disease in patients with primary immunodeficiency diseases. However, no previous review has dealt with the issue of which primary immunodeficiency diseases predispose affected individuals to mycobacterial disease. This information is very useful, not only increasing our understanding of human immunity to mycobacteria, but also for the diagnostic investigation of patients with mycobacteriosis. We review here the medical literature on cases of mycobacterial disease in patients with primary immunodeficiency diseases.