Identification of New Natural CphA Metallo-β-Lactamases CphA4 and CphA5 in Aeromonas veronii and Aeromonas hydrophila Isolates from Municipal Sewage in Central Italy

Two new natural CphA metallo-β-lactamases, the CphA4 and CphA5 enzymes, were identified in water samples from municipal sewage in central Italy. Compared to CphA, the CphA4 and CphA5 enzymes showed numerous point mutations. These enzymes have a narrow spectrum of substrates focused on carbapenems only. CphA5 showed k_cat values about 40-, 12-, and 97-fold higher than those observed for CphA4 versus imipenem, ertapenem, and biapenem, respectively.     

Sphingomyelinase-Like Phosphodiesterase 3b Expression Levels Determine Podocyte Injury Phenotypes in Glomerular Disease

Diabetic kidney disease (DKD) is the most common cause of ESRD in the United States. Podocyte injury is an important feature of DKD that is likely to be caused by circulating factors other than glucose. Soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor found to be elevated in the serum of patients with FSGS and causes podocyte αVβ₃ integrin-dependent migration in vitro. Furthermore, αVβ₃ integrin activation occurs in association with decreased podocyte-specific expression of acid sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) in kidney biopsy specimens from patients with FSGS. However, whether suPAR-dependent αVβ₃ integrin activation occurs in diseases other than FSGS and whether there is a direct link between circulating suPAR levels and SMPDL3b expression in podocytes remain to be established. Our data indicate that serum suPAR levels are also elevated in patients with DKD. However, unlike in FSGS, SMPDL3b expression was increased in glomeruli from patients with DKD and DKD sera-treated human podocytes, where it prevented αVβ₃ integrin activation by its interaction with suPAR and led to increased RhoA activity, rendering podocytes more susceptible to apoptosis. In vivo, inhibition of acid sphingomyelinase reduced proteinuria in experimental DKD but not FSGS, indicating that SMPDL3b expression levels determined the podocyte injury phenotype. These observations suggest that SMPDL3b may be an important modulator of podocyte function by shifting suPAR-mediated podocyte injury from a migratory phenotype to an apoptotic phenotype and that it represents a novel therapeutic glomerular disease target.     

IL-4 in vitro production is upregulated in Alzheimer's disease patients treated with acetylcholinesterase inhibitors

Cytokines appear to be involved in the pathogenesis of Alzheimer's Disease (AD). Their modulation by treatment has been investigated only in a few studies. The aim of our study was to evaluate the effect of acetylcholinesterase inhibitors (AChEI) on Interleukin-4 (IL-4) production in AD patients. IL-4 levels were measured by ELISA on peripheral blood mononuclear cell cultures in the presence or absence of Concanavalin A or Phytohaemagglutinin. Linear regression analysis shows that patients who have been treated, have higher levels of IL-4 independently from age, gender and comorbidity. The increased production of IL-4 in AChEI treated patients might represent an additional mechanism through which AChEI act on AD progression.     

Antibody responses to nasopharyngeal carriage of Streptococcus pneumoniae in adults: a longitudinal household study

BACKGROUND: Natural immunity to Streptococcus pneumoniae is thought to be induced by exposure to S. pneumoniae or cross-reactive antigens. No longitudinal studies of carriage of and immune responses to S. pneumoniae have been conducted using sophisticated immunological laboratory techniques. METHODS: We enrolled 121 families with young children into this study. Nasopharyngeal (NP) swabs were collected monthly for 10 months from all family members and were cultured in a standard fashion. Cultured S. pneumoniae isolates were serotyped. At the beginning (month 0) and end (month 10) of the study, venous blood was collected from family members >18 years old. Serotype-specific antipolysaccharide immunoglobulin G (IgG) and functional antibody and antibodies to pneumolysin, pneumococcal surface protein A (PspA), and pneumococcal surface antigen A (PsaA) were measured in paired serum samples. RESULTS: Levels of anticapsular IgG increased significantly after carriage of serotypes 9V, 14, 18C, 19F, and 23F by an individual or family member. For serotype 14, a higher level of anticapsular IgG at the beginning of the study was associated with reduced odds of carriage (P = .006). There was a small (approximately 20%) but significant increase in titers of antibodies to PsaA and pneumolysin but no change in titers of antibody to PspA. CONCLUSIONS: Adults respond to NP carriage by mounting anticapsular and weak antiprotein antibody responses, and naturally induced anticapsular IgG can prevent carriage.     

Electrophysiological properties of mouse bone marrow c-kit+ cells co-cultured onto neonatal cardiac myocytes

OBJECTIVE: Controversy about hematopoietic stem cells reprogramming into cardiac myocytes is currently supported by positive and negative findings. In fact, some reports have shown the ability of stem cells from the bone marrow (BM) to differentiate into cardiac myocytes and to contribute to myocardium repair, while others have reported the opposite. METHODS: C-kit(+) cells from mouse bone marrow were co-cultured onto neonatal cardiac myocytes. Hematopoietic stem cell-derived cells were analyzed by investigating the expression of cardiac markers and ion channels and by single-cell electrophysiological recordings. RESULTS: Groups of undifferentiated c-kit(+) cells displayed only outward currents. Co-cultured c-kit(+) stem cells on neonatal cardiac myocytes expressed cardiac markers and Na(+) and Ca(2+) voltage-gated ion channels. However, Na(+) and Ca(2+) currents were not detected by electrophysiological patch-clamp recordings even if caffeine and cyclopiazonic acid treatment showed the presence of intracellular calcium stores. This suggests that these channels, although expressed, were not functional and thus do not allow the coupling between excitation and contraction that is typical of cardiac myocytes. Nevertheless, co-cultured cells had a more hyperpolarized resting membrane potential and, at least in a subset of cells, displayed voltage-gated inward rectifier currents and outward currents. Co-cultured c-kit(+)-derived cells were not connected to surrounding cardiac myocytes through gap junctions. To induce a more pronounced differentiation, co-cultured cells were treated with BMP-4 and TGF-beta, two factors that were shown to trigger a cardiac myocyte differentiation pathway in embryonic stem (ES) cells. Even under these conditions, c-kit(+) cells did not differentiate into functionally active cardiac myocytes. However, TGF-beta/BMP-4-treated cells were hyperpolarized and showed and increased inward rectifier current density. CONCLUSIONS: Our study shows that mouse BM hematopoietic stem cells exhibit a limited plasticity to transdifferentiate into cardiac myocytes in culture.     

Frequency of O6‐methylguanine‐DNA methyltransferase promoter methylation in cytological samples from small cell lung cancer

Background. In a phase II study for patients with relapsed small cell lung cancer (SCLC), the administration of Temozolomide, an alkylating agent used in gliomas and anaplastic astrocytoma, showed a effective activity when O⁶‐methylguanine‐DNA methyltransferase (MGMT) gene promoter was methylated. Methods. We tested the feasibility of MGMT promoter status evaluation in small biopsies and cytological specimens routinely processed for diagnostic purposes. We tested samples from 56 patients with SCLC: 30 tissue biopsies, 17 fine‐needle aspiration biopsy, 8 bronchial washing, and 1 was a sputum. Biopsies and fine‐needle aspiration biopsy were fixed in formalin, bronchial washing and sputum in Dubosq Brazil. DNA was extracted after macrodissection of the areas containing the maximum number of cancer cells. MGMT promoter methylation status was assessed by methylation specific PCR. Results. Methylation analysis was obtained in 54 samples (54/56) and failed in two bronchial wash. MGMT promoter was methylated in 35.2% of the cases without any significant difference between histological and cytological samples (37.9% vs. 32%). Conclusion. MGMT promoter methylation is present in SCLC and cytological samples are perfectly adequate for methylation analysis, even if they were taken during routine diagnostic procedures, using different fixative and with low number and percentage of cancer cells. Diagn. Cytopathol. 2015;43:947–952. © 2015 Wiley Periodicals, Inc.     

Accurate estimate of pancreatic T2* values: how to deal with fat infiltration

Purpose

We examined different approaches aimed to deal with the signal fluctuation of pancreatic T2* values due to fat infiltration in order to obtain accurate estimates of iron overload.

Methods

Pancreatic T2* values were assessed in 20 patients (13 females, 37.24 ± 9.12 years) enrolled in the Myocardial Iron Overload in Thalassemia network without and with the application of fat suppression-FS (T2*-NoFS and T2*-FS). T2* values were assessed in three different ways: (1) from the immediate fit (original T2*); (2) discarding the echoes until the achievement of a good visual concordance between the signal and the model (final_vis T2*); (3) eliminating the echoes until the achievement of a fitting error (known) <5% (final_thres T2*).

Results

For the T2*-NoFS sequence the original T2* values were significantly higher than the final_vis T2* values (difference:4.8 ± 6.1 ms; P < 0.0001) and the final_thres T2* values (difference:4.3 ± 6.1 ms; P = 0.006). For the T2*-FS sequence the original T2* values were comparable to final_vis and final_thres T2* values. The original T2*-FS values were significantly different from the original T2*-NoFS values. The final_vis T2*-FS values were comparable to the final_vis T2*-NoFS values and the final_thresh T2*-FS values were comparable to the final_thresh T2*-NoFS values. For both T2*-FS and T2*-NoFS sequences, the final_thres T2* values were not significantly different from the final_vis T2* values and no bias was present.

Conclusions

In the clinical practice, an accurate pancreatic iron overload assessment should be done by applying FS and, when needed, by discarding the TEs until the fitting error goes below 5%.

     

Characterization of persistent and recurrent Cushing’s disease

A case of possible recurrent Cushing’s disease (CD) is presented and data on current definitions of CD remission, persistence, and recurrence are reviewed. While prevailing guidelines recommend the use of serum cortisol in the first post-operative week for defining initial remission and predicting sustained remission, with the use of 24 h urinary free cortisol measurements if serum cortisol values are equivocal, there is debate as to which methods and thresholds best define CD remission and predict successful outcomes. Other methods used to define remission (and hence persistence and recurrence) include restoration of cortisol suppression after dexamethasone and physiologic diurnal cortisol rhythm as measured by midnight salivary free cortisol. However, the number and degree of abnormal test results needed to define recurrence, and the determination of which biochemical test has more significance when there are discrepancies between markers is inconsistent among studies. Further inquiry is warranted to examine if patients in apparent CD remission who have subtle hypothalamic pituitary adrenal (HPA) axis abnormalities represent distinctive remission subtypes versus mild or early recurrence. Additional investigation could also explore the degree to which these HPA axis abnormalities, such as alterations in cortisol circadian rhythm or partial resistance to dexamethasone, are associated with persistence of CD morbidities, including neuropsychiatric impairments, alterations in body composition, and cardiovascular risk.