Characterization of the large-conductance Ca-activated K channel in myocytes of rat saphenous artery

We used the patch-clamp method to characterize the BK channel in freshly isolated myocytes from the saphenous branch of the rat femoral artery. Single-channel recordings revealed that the BK channel had a conductance of 187 pS in symmetrical 150 mM KCl, was blocked by external tetraethylammonium (TEA) with a KD(TEA) of approx. 300 microM at +40 mV, and by submicromolar charybdotoxin (CTX). The sensitivity of the BK channel to Ca was especially high (KD(ca) approx. 0.1 microM at +60 mV) compared to skeletal muscle and neuronal tissues. We also investigated the macroscopic K current, which under certain conditions is essentially sustained by BK channels. This conclusion is based on the findings that the macroscopic current activated upon depolarization follows a single exponential time course and is virtually fully blocked by 100 nM CTX and 5 mM external TEA. We made use of this occurrence to assess the voltage and Ca dependence of the macroscopic BK current. In intact myocytes, the BK channel showed a strong and voltage-dependent reduction of the outward current (62% at +40 mV), most likely due to block by intracellular Ba and polyamines. The results obtained from macroscopic and unitary current indicate that approx. 2.5% of the BK channels are active under physiological conditions, sustaining approx. 20 pA of outward current. Given the high input resistance of these cells, few BK channels are required to open in order to cause a significant membrane hyperpolarization, and thus function to limit the contraction resulting from acute increases in intravascular pressure, or in response to hypertensive pathologies.     

Antibiotic Use in Crohn’s Disease

On the assumption that bacteria in the gut may be a cause of symptoms and/or complications of Crohn's disease, various antibiotics are efficaciously employed in some affected patients. However, we do not know exactly why and how they are helpful. A possible explanation is that one or several bacterial species may have a primary role in the aetiology of Crohn's disease, but this is not supported by the data in our possession. Another hypothesis is that intestinal bacteria may cause flare-up of the disorder, either by inducing intestinal lesions or by an interaction with the immune system, but we know today that specific pathogens can cause flares only in a minority of cases. On the contrary, there is considerable evidence that the intestinal microflora and its products may amplify and perpetuate inflammation in Crohn's disease. Despite the fact that few controlled trials have been conducted, and have shown inconclusive results, antibiotics are widely employed for improving symptoms and for inducing remission of active phases. At present, a combination of metronidazole and ciprofloxacin, active against many enteric bacteria, has proved to be effective in the treatment of Crohn's disease complications. This therapy also seems to be effective in acute flares as an alternative to, or in combination with, corticosteroids.     

Effects of HDL3 on the expression of matrix-degrading proteases in human endothelial cells

Modified lipoproteins have been suggested to modulate the expression of matrix-degrading proteases in the vascular wall. Since oxidized high density lipoprotein (HDL) has been found in atheromatous plaques and receptors for modified HDL are present on endothelial cells, we investigated the role of native and oxidized HDL3 on the expression of 35 proteases and their inhibitors in human endothelial cells using microarray analysis. Matrix metalloproteinase (MMP)-1, -2, -10, -13 and -14, tissue inhibitor of MMP (TIMP)-1, -2 and -3, cathepsin B and D, and cystatin C were expressed under basal conditions, of which MMP-10 and cystatin C expression have not been described before in endothelial cells. Native HDL3 increased MMP-1 and MMP-14 expression and decreased MMP-13 expression, whereas oxidized HDL3 increased PAI-1 and MMP-1 expression. The expression pattern was confirmed by quantitative real-time PCR. In summary, a large repertoire of matrix-degrading proteases is expressed in endothelial cells, an expression that can be modulated by native and oxidized HDL3.     

Dynamic responses of oxygen uptake at the onset and end of moderate and heavy exercise in trained subjects.

Inconsistencies about dynamic asymmetry between the on- and off-transient responses in VO2 are found in the literature. Therefore the purpose of this study was to examine VO2 on- and off-transients during moderate- and heavy-intensity cycling exercise in trained subjects. Ten men underwent an initial incremental test for the estimation of ventilatory threshold (VT) and, on different days, two bouts of square-wave exercise at moderate (VT) intensities. VO2 kinetics in exercise and recovery were better described by a single exponential model (VT). For moderate exercise, we found a symmetry of VO2 kinetics between the on- and off-transients (i.e., fundamental component), consistent with a system manifesting linear control dynamics. For heavy exercise, a slow component superimposed on the fundamental phase was expressed in both the exercise and recovery, with similar parameter estimates. But the on-transient values of the time constant were appreciably faster than the associated off-transient, and independent of the work rate imposed (VT). Our results do not support a dynamically linear system model of VO2 during cycling exercise in the heavy-intensity domain.     

Gene Therapy in Infants with Severe Combined Immunodeficiency

Severe combined immunodeficiencies (SCID) are rare disorders that represent paediatric medical emergencies, as the outcome for affected patients can easily be fatal unless proper treatment is performed. The only curative treatment for SCID is reconstitution of the patient's immunity. For more than 30 years, allogeneic bone marrow transplantation (BMT) has been extremely successful for SCID. However, BMT often results in only incomplete restoration of B cell function in treated patients, especially when haploidentical donors are used. In addition, BMT can be associated with severe complications such as graft-versus-host disease (GVHD). Alternative forms of therapy for SCID are therefore desirable. Genetic correction of peripheral T lymphocytes and/or haematopoietic stem cells (HSCs) by retrovirally mediated gene transfer has been attempted for patients with SCID due to adenosine deaminase deficiency, the first genetic disease targeted in clinical gene therapy trials with very limited success, overall. After these pioneer trials, recent progress has led to significant improvement of gene transfer techniques and better understanding of HSC biology which has culminated in the recent success of a gene therapy trial for patients affected with X-linked SCID (X-SCID). In this trial, patients with X-SCID received autologous bone marrow stem/progenitor cells which had been retrovirally transduced with a therapeutic gene. Based on the current follow-up, the overall efficacy of this gene therapy procedure is to be considered similar to or even better than that achievable by allogeneic BMT, because patients were not exposed to the risks of GVHD. Although these exciting results have clearly demonstrated that gene therapy is a feasible therapeutic option for X-SCID, they have also raised important questions regarding the long-term outcome of this experimental procedure and the possibility of translating this success into applications for other forms of SCID.     

Therapeutic and diagnostic applications of minor histocompatibility antigen HA-1 and HA-2 disparities in allogeneic hematopoietic stem cell transplantation: a survey of different populations.

Minor histocompatibility antigens (mHags) HA-1 and HA-2 are encoded by biallelic loci, with immunogenic variants, HA-1H and HA-2V, which induce strong HLA-A2-restricted alloreactive T-cell responses, and nonimmunogenic counterparts, HA-1R and HA-2M, which represent functional null alleles that are poorly presented by HLA class I molecules. HA-1 and HA-2 are potential targets of selective graft-versus-leukemia and graft-versus-tumor reactivity after allogeneic hematopoietic stem cell transplantation (HSCT); however, these applications are restricted to a limited number of patients. Here, we show that a far more frequent application of HA-1 and HA-2 disparity relies on their use as markers for the state of host chimerism after allogeneic HSCT. We have determined allelic frequencies of 29.3% and 70.7% for HA-1H and HA-1R, respectively, and of 83.7% and 16.3% for HA-2V and HA-2M, respectively, in >200 healthy individuals from northern Italy. Similar frequencies were observed in nearly 100 patients affected by hematologic malignancies or solid tumors, thus showing that HA-1 and HA-2 variability are not associated with the presence of cancer. On the basis of these data, we predict that HA-1 and HA-2 can be used in 32.8% and 23.5% of Italian transplant patients, respectively, as markers for the state of host chimerism, whereas exploitation of disparity for these mHags for targeted immunotherapy will be possible in 10.7% and 1.1% of Italian patients, respectively. Retrospective HA-2 typing of bone marrow aspirates obtained from a patient during complete remission or recurrence of acute myeloid leukemia after haploidentical HSCT showed the feasibility of using HA-2 as a surrogate marker for disease monitoring. Because of an apparent north-south gradient for HA-1 allelic frequencies, with higher frequencies for the HA-1H variant reported in white populations from Southern Europe as compared with Northern Europe and North America, the diagnostic applicability of HA-1 disparity will be slightly more frequent in transplant patients from the north. Taken together, our data show that determination of HA-1 and HA-2 variability can be an important parameter for the selection of allogeneic stem cell donors, in particular for patients affected by hematologic malignancies without a tumor-specific molecular marker.     

Mast cell populations in the chick embryo lung and their response to compound 48/80 and dexamethasone

Summary Two mast cell populations, connective tissue mast cells (CTMCs) and mucosal mast cells, (MMCs) containing different proteoglycans in their granules, can be distinguished in several animal species by means of histochemical methods. In this study we documented the presence of these two types of mast cell in the chick embryo lung, from the 15th incubation day for the MMCs, and from the 18th incubation day for the CTMCs. Lungs of embryos treated with compound 48/ 80, which produces degranulation of the CTMCs, showed a decrease in the number of this type of mast cell and an unchanged number of MMCs. In the lungs of embryos treated with dexamethasone, which degranulates MMCs, a reduction in the number of these cells and an unchanged number of the CTMs were found.     

Cryptosporidium parvum-specific CD4 Th1 cells from sensitized donors responding to both fractionated and recombinant antigenic proteins

T-cell-mediated immunity plays a central role in the host response to Cryptosporidium parvum. Human T-cell clones (TCC) were isolated from peripheral blood mononuclear cells of five healthy donors with prior cryptosporidiosis by use of a C. parvum crude extract, two antigen fractions obtained by ion-exchange chromatography (IEC1 and IEC2), and two recombinant peptides (SA35 and SA40) from C. parvum sporozoites. The T-cell lines derived from the one recently infected donor had a higher proportion (26 to 38%) of T cells exhibiting the gamma/delta T-cell receptor (gamma/delta-TCR) than those from donors who had recovered from cryptosporidiosis several years earlier, suggesting that the gamma/delta T-cell population is involved in the early stage of the infection. The specific TCC had the alpha/beta-TCR, had the phenotype CD45RO(+) CD4(+) CD8(-), and were characterized by either hyperproduction of gamma interferon (IFN-gamma) alone, with a Th1 profile, or IFN-gamma hyperproduction together with interleukin-4 (IL-4) or IL-5 production, with a Th0 profile. SA35, SA40, IEC1, and IEC2 may be considered good targets of the cellular response against C. parvum and may play a role in maintaining the T-cell-mediated memory response to this parasite. Furthermore, the SA35 and SA40 peptides may be regarded as immunodominant antigens involved in the maintenance of the T-cell response in healthy C. parvum-sensitized persons.     

Severe congenital muscular dystrophy in a Mexican family with a new nonsense mutation (R2578X) in the laminin alpha-2 gene

The congenital muscular dystrophies (CMDs) are a heterogeneous group of autosomal recessive disorders. Approximately one half of cases diagnosed with classic CMD show primary deficiency of the laminin alpha2 chain of merosin. Complete absence of this protein is usually associated with a severe phenotype characterized by drastic muscle weakness and characteristic changes in white matter in cerebral magnetic resonance imaging (MRI). Here we report an 8-month-old Mexican female infant, from a consanguineous family, with classical CMD. Serum creatine kinase was elevated, muscle biopsy showed dystrophic changes, and there were abnormalities in brain MRI. Immunofluorescence analysis demonstrated the complete absence of laminin alpha2. In contrast, expression of alpha-, beta-, gamma-, and delta-sarcoglycans and dystrophin, all components of the dystrophin-glycoprotein complex, appeared normal. A homozygous C long right arrow T substitution at position 7781 that generated a stop codon in the G domain of the protein was identified by mutation analysis of the laminin alpha2 gene ( LAMA2). Sequence analysis on available DNA samples of the family showed that parents and other relatives were carriers of the mutation.