A Novel Mutation in a Critical Region for the Methyl Donor Binding in DNMT3B Causes Immunodeficiency,Centromeric Instability,and Facial Anomalies Syndrome (ICF)

Purpose

Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is an extremely rare autosomal recessive disease. The immune phenotype is characterized by hypogammaglobulinemia in the presence of B cells. T cell lymphopenia also develops in some patients. We sought to further investigate the immune defect in an ICF patient with a novel missense mutation in DNMT3B and a severe phenotype.

Methods

Patient lymphocytes were examined for subset counts, immunoglobulin levels, T and B cell de novo production (via excision circles) and receptor repertoire diversity. Mutated DNMT3B protein structure was modeled to assess the effect of a mutation located outside of the catalytic region on protein function.

Results

A novel homozygous missense mutation, Ala585Thr, was found in DNMT3B. The patient had decreased B cell counts with hypogammaglobulinemia, and normal T cell counts. CD4⁺ T cells decreased over time, leading to an inversion of the CD4⁺ to CD8⁺ ratio. Excision circle copy numbers were normal, signifying normal de novo lymphocyte production, but the ratio between naïve and total B cells was low, indicating decreased in vivo B cell replication. T and B cell receptor repertoires displayed normal diversity. Computerized modeling of the mutated Ala585 residue suggested reduced thermostability, possibly affecting the enzyme kinetics.

Conclusions

Our results highlight the existence of a T cell defect that develops over time in ICF patient, in addition to the known B cell dysfunction. With intravenous immunoglobulin (IVIG) treatment ameliorating the B cell defect, the extent of CD4⁺ lymphopenia may determine the severity of ICF immunodeficiency.

     

Proteases, extracellular matrix, and cancer: a workshop of the path B study section

The role of the extracellular matrix (ECM) in the tumor microenvironment is not limited to being a barrier against tumor invasion. The ECM is a reservoir of cell binding proteins and growth factors that affect tumor cell behavior. It is also substantially modified by proteases produced by tumor cells or stroma cells. As a result of the activity of these proteases, cell-cell and cell-ECM interactions are altered, new biologically active ECM molecules are generated, and the bioavailability and activity of many growth factors, growth factor receptors, and cytokines are modified. ECM-degrading proteases also play a critical role in angiogenesis, where they can act as positive as well as negative regulators of endothelial cell proliferation and vascular morphogenesis. This review article summarizes some of the most relevant findings made over the recent years that were discussed at a workshop organized by the Path B Study Section of the National Institutes of Health in October 2002.     

Alterations in galectin-3 expression and distribution correlate with breast cancer progression: functional analysis of galectin-3 in breast epithelial-endothelial interactions

To define the role of galectin-3 in breast cancer progression, we have used a novel three-dimensional co-culture system that recapitulates in vivo reciprocal functional breast epithelial-endothelial cell-cell and cell-matrix interactions, and examined the expression of galectin-3 mRNA and protein in human breast tumors and xenografts. Galectin-3 is required for the stabilization of epithelial-endothelial interaction networks because immunoneutralization with galectin-3 antibodies abolishes the interactions in a dose-dependent manner. Co-culture of epithelial cells with endothelial cells results in increase in levels of secreted galectin-3 and presence of proteolytically processed form of galectin-3 in the conditioned media. In contrast, intracellular galectin-3 predominantly exists in the intact form. This difference in sensitivity to proteolytic processing of secreted versus intracellular galectin-3 probably arises from differences in accessibility of protease-sensitive sites, levels, and/or type of activated protease(s), and may be indicative of different functional roles for intact and processed galectin-3. To determine whether the proteolytically cleaved galectin-3 retains its ability to bind to endothelial cells, binding assays were performed with the full-length and matrix metallopeoteinase-2-cleaved recombinant galectin-3. Although a dose-dependent increase in binding to human umbilical vein endothelial cells was observed with both full-length and cleaved galectin-3, proteolytically cleaved galectin-3 displayed approximately 20-fold higher affinity for human umbilical vein endothelial cells as compared to the full-length protein. Examination of galectin-3 expression in breast tumors and xenografts revealed elevated levels of galectin-3 mRNA and protein in the luminal epithelial cells of normal and benign ducts, down-regulation in early grades of ductal carcinoma in situ (DCIS), and re-expression in peripheral tumor cells as DCIS lesions progressed to comedo-DCIS and invasive carcinomas. These data suggest that galectin-3 expression is associated with specific morphological precursor subtypes of breast cancer and undergoes a transitional shift in expression from luminal to peripheral cells as tumors progressed to comedo-DCIS or invasive carcinomas. Such a localized expression of galectin-3 in cancer cells proximal to the stroma could lead to increased invasive potential by inducing novel or better interactions with the stromal counterparts.     

Mixed functional characteristics correlating with TCR‐ligand koff‐rate of MHC‐tetramer reactive T cells within the naive T‐cell repertoire

The low frequency of antigen‐specific naïve T cells has challenged numerous laboratories to develop various techniques to study the naïve T‐cell repertoire. Here, we combine the generation of naïve repertoire‐derived antigen‐specific T‐cell lines based on MHC‐tetramer staining and magnetic‐bead enrichment with in‐depth functional assessment of the isolated T cells. Cytomegalovirus (CMV) specific T‐cell lines were generated from seronegative individuals. Generated T‐cell lines consisted of a variety of immunodominant CMV‐epitope‐specific oligoclonal T‐cell populations restricted to various HLA‐molecules (HLA‐A1, A2, B7, B8, and B40), and the functional and structural avidity of the CMV‐specific T cells was studied. Although all CMV‐specific T cells were isolated based on their reactivity toward a specific peptide‐MHC complex, we observed a large variation in the functional avidity of the MHC‐tetramer positive T‐cell populations, which correlated with the structural avidity measured by the recently developed Streptamer k_off‐rate assay. Our data demonstrate that MHC‐tetramer staining is not always predictive for specific T‐cell reactivity, and challenge the sole use of MHC‐tetramers as an indication of the peripheral T‐cell repertoire, independent of the analysis of functional activity or structural avidity parameters.     

The inhibitory receptor CD300a is essential for neutrophil-mediated clearance of urinary tract infection in mice

Neutrophils play a crucial role in immune defense against and clearance of uropathogenic Escherichia coli (UPEC)-mediated urinary tract infection, the most common bacterial infection in healthy humans. CD300a is an inhibitory receptor that binds phosphatidylserine and phosphatidylethanolamine, presented on the membranes of apoptotic cells. CD300a binding to phosphatidylserine and phosphatidylethanolamine, also known as the “eat me” signal, mediates immune tolerance to dying cells. Here, we demonstrate for the first time that CD300a plays an important role in the neutrophil-mediated immune response to UPEC-induced urinary tract infection. We show that CD300a-deficient neutrophils have impaired phagocytic abilities and despite their increased accumulation at the site of infection, they are unable to reduce bacterial burden in the bladder, which results in significant exacerbation of infection and worse host outcome. Finally, we demonstrate that UPEC's pore forming toxin α-hemolysin induces upregulation of the CD300a ligand on infected bladder epithelial cells, signaling to neutrophils to be cleared.     

The effect of erythropoietin therapy and hemoglobin levels on the immune response to Engerix-B vaccination in chronic kidney disease

BACKGROUND: Patients undergoing chronic hemodialysis have an increased risk of acquiring hepatitis B infection. Only 43-66% of dialysis patients develop effective anti-HBs titers after vaccination. AIM: To evaluate the effect of recombinant erythropoietin (rEPO) therapy and basal hemoglobin levels on the outcome of the immune response to four doses of IM 40 microg Engerix-B vaccination in hemodialysis and chronic kidney disease (CKD) patients before starting replacement therapy. SUBJECTS AND METHODS: One hundred and three patients were included in the study: 34 hemodialysis patients treated with rEPO (Group A), 36 predialytic patients who did not treated with rEPO (Group B) and 33 predialytic patients treated with rEPO (Group C). Plasma creatinine in predialytic patients was 2-7 mg/dL. All patients' HBsAg and anti-HBs antibodies were negative. Patients were immunized with IM 40 microg Engerix-B at 0, 1, 3, and 6 months. Anti-HBs titers were measured at 7th month. RESULTS: Eighty seven point one percent of patients from group C developed protective anti-HBs titers compared with 69.4% from group B and 44.1% from group A (p = 0.001). Patients from all groups with baseline hemoglobin levels above 11 gr/dL developed protective anti-HBs titers significantly more than patients with baseline hemoglobin levels below 11 gr/dL (p < 0.05). CONCLUSION: Predialytic patients treated with rEPO and with hemoglobin levels higher than 11 gr/dL had significantly better immune response outcomes to Engerix-B vaccination. Immunization against hepatitis B infection should be considered at early stages of CKD prior to the deterioration in kidney functions and the development of renal anemia.     

The relationship of chronic pain with and without comorbid psychiatric disorder to sleep disturbance and health care utilization: results from the Israel National Health Survey

Objective

Chronic pain is associated with health problems including sleep difficulties and increased medical utilization. Because chronic pain is frequently comorbid with psychiatric disorders, it is unclear to what degree chronic pain itself is associated with these problems. In a large population sample, we examined the relationship between chronic pain, both alone and comorbid with psychiatric disorders, with sleep disturbance and increased medical utilization.

Methods

We analyzed data from the Israel National Health Survey (INHS) conducted in 2003-2004 on a representative sample (N=4859) of the adult Israeli population. Data were collected in face-to-face interviews using the Composite International Diagnostic Interview. Statistical analyses were performed using multinomial logistic regression models.

Results

Past year chronic pain was reported by 29.9% of all study participants (n=1453). Psychiatric disorders were more common among participants with chronic pain; adjusted odds ratios were 2.23 (95% CI 1.49-3.36) for depressive disorders and 2.94 (95% CI 2.08-4.17) for anxiety disorders. Associations of chronic pain and psychiatric disorders were stronger in men. Chronic pain was associated with both sleep problems and increased health care utilization even for individuals with no psychiatric comorbidity. Sleep difficulties but not health care utilization rates were more pronounced in the comorbid group compared to the chronic pain only group.

Conclusion

Chronic pain was associated with sleep problems and increased health care utilization in this sample, independent of psychiatric comorbidity. Sleep problems were significantly greater in the comorbid vs. non-comorbid group. In contrast, associations of pain with health care utilization were largely independent of psychiatric comorbidity.     

Is the diagnostic yield of prostate needle biopsies affected by prostate volume?

ObjectivesTo determine the effect of prostate volume on the diagnostic yield of prostate biopsies.Materials and methods155 consecutive patients underwent 12-core transrectal ultrasound guided needle biopsies. Data were collected prospectively on age, serum PSA, digital rectal examination (DRE), previous prostate biopsies, prostate volume and pathologic result. Univariate and multivariate logistic regressions were undertaken to determine the effect of prostate volume on the risk for a positive biopsy.Results45 patients (29%) were diagnosed with cancer. The median patient age was 63 (range 48–82) years, the median PSA level was 6.7 ng/ml (0.5–156 ng/ml), and the median prostate volume was 57 ml (16–273 ml). 42 patients (27%) had an abnormal DRE and 51 (33%) had undergone previous prostate biopsies. Positive biopsy rates were 39%, 33%, and 14% for prostate volume below 46 ml, between 45 and 73 ml, and above 72 ml, respectively. Univariate analysis showed that age, serum PSA, DRE and prostate volume were all associated with a positive biopsy. Multivariate analysis adjusted for age, PSA and DRE showed a significant risk increase for a positive biopsy in smaller prostates. (OR = 5.6 95% CI 1.75–17.89; and 8.86 95% CI 2.72–28.82, for prostate volume between 45 and 72 ml and below 45 ml, respectively).ConclusionThe diagnostic yield of prostate biopsies is significantly lower in large prostates. As the result the standard 12-core biopsy may be insufficient for the diagnosis of cancer in large prostates.     

Cytoscopic light test to aid in the differentiation of high-grade pelvic organ prolapse

It remains quite difficult to distinguish a high-grade cystocele from an enterocele or high rectocele on the basis of physical examination findings alone. We have employed the use of a cystoscopic light test during preoperative or intraoperative endoscopy to assist in differentiating these entities.     

Laparoscopic extraperitoneal inguinal hernia repair with spinal anesthesia and nitrous oxide insufflation

Background: Laparoscopic repair of inguinal hernia is traditionally performed under general anesthesia mainly because of the adverse effects that carbon dioxide pneumoperitoneum has on awake patients. Since a mandatory use of general anesthesia for all hernia repairs is questionable, the feasibility of laparoscopic extraperitoneal herniorraphy using spinal anesthesia combined with nitrous oxide insufflation was investigated. Methods: Over a 4-month period, February to May 1998, we performed 35 consecutive total extraperitoneal inguinal hernia procedures (24 unilateral, 11 bilateral) using spinal anesthesia and nitrous oxide extraperitoneal gas. Data on operative findings, self-reported operative and postoperative pain and discomfort (visual analog pain scale), procedure-related hemodynamics, and complications were collected prospectively. Results: All 35 procedures were completed laparoscopically without the need to convert to general anesthesia. Mean operative time was 39 ± 7 min for unilateral hernia and 65 ± 10 min for bilateral hernia. Incidental peritoneal tears occurred in 22 patients (63%) resulting in nitrous oxide pneumoperitoneum, which was well tolerated. The patients remained hemodynamically stable throughout the procedure, and operative conditions and visibility were excellent. Complications at a mean of 4 months after the procedure included seven uninfected seromas (20%), three patients with transient testicular pain, and one (3%) recurrence. Conclusions: Laparoscopic total extraperitoneal hernia repair can be safely and comfortably performed using spinal anesthesia with extraperitoneal nitrous oxide insufflation gas. This method provides a good alternative to general anesthesia. Received: 17 February 1999/Accepted: 1 July 1999