Petru Cianga

IDENTIFICATION AND FUNCTION OF NEONATAL FC RECEPTOR IN MAMMARY GLAND OF LACTATING MICE

In addition to its proposed function in regulating serum IgG levels, the MHC class I‐related neonatal Fc receptor (FcRn) is known to play a role in IgG transfer across rodent yolk sac and neonatal intestine. In contrast to humans, for which transplacental transfer of IgG appears to be the only mechanism of maternal IgG delivery, the transmission of IgG in mice occurs both antenatally (yolk sac) and neonatally (transport from mothers milk across intestinal epithelial cells). In the current study, a possible role for FcRn in regulating IgG transfer into milk has been investigated. FcRn has been shown to be present in functional form in the mammary gland of lactating mice, and is localized to the epithelial cells of the acini. Analysis of the transfer of Fc fragments and IgG which have different affinities for FcRn indicate that, unexpectedly, these proteins are transferred in inverse correlation with their binding affinity for FcRn. Thus, in the lactating mammary gland FcRn appears to play a role in recycling IgG in a mode that may have relevance to FcRn trafficking during the maintenance of constant serum IgG levels.

Comparative studies of rat IgG to further delineate the Fc:FcRn interaction site.

Recent data have indicated that the MHC class I‐related receptor, FcRn, regulates the half‐lives of serum IgG in addition to its known role in transferring IgG from mother to young. In the current study, the activity of rat IgG (rIgG) isotypes in FcRn‐mediated functions has been analyzed. The serum half‐life and maternofetal transfer in mice decreased in the order rIgG2a > rIgG1 > rIgG2c > rIgG2b. This decrease in activity correlates well with reduced binding affinity for soluble mouse FcRn, and site‐directed mutagenesis of a recombinant Fc‐hinge fragment has been used to investigate the molecular basis for the differences in activities of the rIgG. Analysis of the serum half‐lives of the mutated Fc‐hinge fragments demonstrated that, in addition to Ile253, His310, His435 and His436 that were identified in earlier studies, amino acids at positions 257, 307 and 309 play a role in building the FcRn interaction site of IgG. The study also excludes the involvement of amino acids in a fourth loop located at the CH2‐CH3 domain interface that encompasses residues 386 – 387 in FcRn binding. Sequence differences at positions 257, 307 and 309 between rIgG most likely account for the reduced affinity of rIgG2b and IgG2c relative to rIgG1 and rIgG2a for binding to FcRn.

Nonclassical major histocompatibility complex I–like Fc neonatal receptor (FcRn) expression in neonatal human tissues

The neonatal Fc receptor (FcRn) was demonstrated to play a role both in the recycling and thus the protection of immunoglobulin G (IgG) from catabolism and in the maternal-fetal transfer of IgG. The expression of this particular receptor was evidenced in a variety of cell types, but the endothelial cell was considered the main cell able to perform both recycling and IgG catabolism. Based on preliminary data obtained in adult human mammary glands and skin, this study focused on a number of neonatal human tissues, targeting FcRn expression mainly in epithelial versus endothelial cells. Our results demonstrate that in most of the investigated tissues, the neonatal Fc receptor is not detectable in the endothelial cells lining the capillaries, whereas most epithelial cells are positive. We could also observe the receptors expression in most macrophages, smooth muscle cells, and neurons. Taken together, these data suggest that the main sites of IgG catabolism might in fact be other than endothelial cells in human neonates.

Immunohistochemical analysis of steroid receptors, proliferation markers, apoptosis related molecules, and gelatinases in non-neoplastic and neoplastic endometrium

Endometrioid endometrial carcinoma developed from endometrial hyperplasia is associated with anomalies of proliferation, apoptosis, and matrix metalloproteinase (MMP) expression. Our study was designed to investigate steroid receptor (ER, PR) expression and its correlation with proliferative activity (PCNA), apoptosis (Fas, FasL, Bcl-2, Bax, and p53), gelatinases (MMP-2 and MMP-9) and their tissue specific inhibitor (TIMP-1 and TIMP-2) immunoexpression in endometrial carcinogenesis. A total of 38 cases were investigated, 10 non-neoplastic, 11 hyperplastic, and 17 carcinomatous endometria. Immunolabeling showed a higher expression of steroid receptors in hyperplasia and carcinoma than in non-neoplastic endometria and an ER/PR imbalance in carcinoma. The epithelial component of endometrial carcinomas had the highest proliferative index. Bcl-2 had a stronger expression in hyperplasia and carcinoma compared to non-neoplastic endometria and stromal tissue. The Bcl-2/Bax ratio was lower in endometrial carcinoma. Fas and FasL expression was stronger in hyperplasia and furthermore in carcinoma. p53 expression was progressively stronger along the sequence non-neoplastic endometrial to hyperplasia-carcinoma. Both types of investigated MMPs showed an increased expression in neoplastic endometria reaching a maximum level in carcinomas. MMP-9 immunostaining could be correlated to myometrial invasion. TIMP-1 decreased and TIMP-2 increased in expression from non-neoplastic endometria to hyperplastic and carcinomatous endometrial, respectively. Our study demonstrates that coordinated anomalies of steroid receptors, apoptosis and invasiveness factors are already present in hyperplasia as cumulative steps along the way to malignant transformation and that a complex MMP-2, MMP-9, TIMP-2/TIMP-1 imbalance seems to be responsible for the endometrial proliferation.

The neonatal Fc receptor (FcRn) expression in the human skin

The neonatal Fc receptor, FcRn, has a wide expression within the human body, including adult cells; however, it is not ubiquitously expressed (reviewed in [2]). The receptor is involved both in IgG transcytosis as well as recycling, being able to bind both free and antigen complexed IgG molecules (reviewed in [4]). Cauza et al. [1] have shown that the neonatal receptor is present in the human epidermal keratinocytes, using both biopsies and cultivated keratinocytes. Furthermore, they were able to demonstrate that most of the FcRn molecules are expressed intracellularly, in acidified vesicles [1], and that the receptor is functional within these cells. However, the FcRn role in keratinocytes in terms of recycling, transcytosis, or perhaps both remains speculative. In this paper, we show by immunohistochemistry that FcRn expression in human skin is not restricted only to keratinocytes but has a wider expression. Tissues were obtained from five human cadavers (harvested in an interval of 24 h from the time of death), and a surgical biopsy was performed for diagnostic purposes (nevus). Immunohistochemistry was done on consecutive skin sections as previously described [2]. The epithelial cells were evidenced with an antihuman cytokeratins antibody (clone MNF116, Dako). As expected, the antibody stained the epithelial cells of the hair follicles and sebaceous glands and the keratinocytes (data not shown). The FcRn-staining pattern proved to be more complex. The epithelial structures, evidenced by the cytokeratins targeting, proved to be positive as well for the Fc neonatal receptor (Fig. 1). Furthermore, we were able to show that the cytokeratinnegative, S-100-positive melanocytes (data not shown) [3] express also this receptor (Fig. 2). Histiocytes and dendritic cells were also shown to be FcRn positive by others [1, 5]. The characterization of the exact nature of the cells scattered throughout the investigated tissues was beyond the goals of this study. However, we can speculate that the isolated FcRn-positive cells in our skin sections are histiocytes and dendritic cells. The presence of FcRn in endothelial cells was investigated by labeling sequential sections with anti-FcRn and respectively anti-CD34 II (clone QBEnd, Dako) antibodies, as well as double labeling with HRP/DAB (brown) for FcRn, and Alkaline Phosphatase/FastRed (Dako, Denmark) (red) for CD34 II. Consistent with the results obtained on human mammary gland sections [2], blood vessels are either negative for the neonatal Fc receptor or express this molecule at a level that is undetectable by the immunohistochemical method (Fig. 3). IgG is the main antibody isotype in the extravascular body spaces. Hence, the wide expression of the neonatal Fc receptor throughout the organism, including in various cell types of the skin, does not come as a surprise. Although the receptor was proven to be functional in keratinocytes [1], the FcRn function (recycling, transcytosis, or both) in the skin structures remains to be elucidated. On the other hand, Virchows Arch (2007) 451:859–860 DOI 10.1007/s00428-007-0467-7

A Role for the Region Encompassing the c″ Strand of a TCR Vα Domain in T Cell Activation Events

The distinct strand topology of TCR Vα domains results in a flatter surface in the region encompassing the c″ strand than the corresponding region in Ig V domains. In the current study a possible role for this region in T cell activation has been investigated by inserting a potential glycosylation site at Vα residue 82. This residue is in proximity to the c″ strand and distal to the putative interaction site for cognate peptide:MHC ligand. An additional N-linked carbohydrate at this position would create a protrusion on the Vα domain surface, and this may interfere with TCR aggregation and/or recruitment of signaling molecules. The modified TCR has been expressed in transfected T cells, and the phenotype following stimulation has been compared with that of cells expressing the wild-type TCR. The mutation has significant effects on activation-induced cell death and TCR internalization, but, unexpectedly, does not affect IL-2 secretion. Furthermore, analyses with tetrameric, peptide:MHC class II complexes suggest that the mutation decreases the ability of the TCR to aggregate into a configuration compatible with avid binding by these multivalent ligands.

Saliva leukocytes rather than saliva epithelial cells represent the main source of DNA

Abstract Introduction. Several alternative methods to peripheral blood DNA extraction have been implemented so far. Saliva seems to represent a very advantageous type of sample, easy to harvest and able to generate DNA yields comparable to those extracted from blood mononuclear cells. Material and methods. 8 patients suspected of ankylosing spondylitis, 9 patients with various hematological malignancies, displaying post-chemotherapy leucopenia and 30 healthy volunteers were included in our study. DNA was extracted with various commercially available kits and used for HLA typing either by PCR amplification, or by PCR followed by hybridization. Results. Our data regarding HLA typing support already published results regarding the good DNA quality that allows its use in various molecular biology techniques. However, when attempting to use saliva from immunosuppressed patients for DNA extraction we have generated very low yields, comparable again with the ones obtained from peripheral blood. Flow cytometry and immunocytochemistry investigations confirmed the low number of leukocytes present in the saliva of these patients, while the number of epithelial cells was virtually unchanged. Conclusions. The main source of saliva DNA seems to be represented by leukocytes present in this fluid and not by the epithelial cells. Under these circumstances, for immunosuppressed patients saliva cannot represent an alternative to blood when attempting DNA extraction.

FLT-3 ITD Positive Acute Basophilic Leukemia with Rare Complex Karyotype Presenting with Acute Respiratory Failure: Case Report

Abstract Background: Acute basophilic leukemia is a rare subtype of acute myeloid leukemia, as categorized by the 2008 World Health Organization classification of myeloid neoplasms. Acute basophilic leukemia diagnosis requires thorough morphological, cytochemical, immunophenotypic, molecular, and cytogenetic studies and exclusion of other hematological neoplasms associating basophilia. The disease course is defined by histamine driven, occasionally life-threatening respiratory, cardiovascular, cutaneous or digestive complications, as well as primary refractoriness to standard therapy. Clinical presentation: We herein report a case of a 63-year-old asthmatic female patient diagnosed with acute basophilic leukemia, associated with previously unpublished cytogenetic features and FLT-3 ITD mutation, pulmonary leukostasis and spontaneous pulmonary capillary leak syndrome, which worsened immediately following chemotherapy initiation. Respiratory complications were successfully managed, but recrudesced upon emergence of refractory disease and were ultimately fatal. We highlight the likelihood of pulmonary complications induced by basophil degranulation and tumor lysis in hypercellular acute basophilic leukemia and the potential benefit of histamine receptor blockade in this setting.

Prognostic values of cardiac biomarkers screening in dialysis patients

Patients with chronic kidney disease (CKD) have an increased cardiovascular (CV) risk and a higher mortality rate. This case report aims to reveal the prognostic value of cardiac biomarkers in patients with CKD stage 5 on dialysis. Several biomarkers have proven their utility for early detection of CV risk in dialysis patients. Most promising biomarkers are: N-terminal pro–B-type (NT-proBNP), high sensitivity cardiac troponin T (Hs–cTnT), Cardiotrophin-1 (CT-1) and Galectin-3 (GAL-3). We report the case of a 44 year-old woman with end stage renal disease on dialysis without any cardiac pathology, but who had, at screening evaluation, high values of cardiac biomarkers, with increasing levels of NT-proBNP at serial determination; Hs-cTnT was constant. Recently, our patient was admitted in cardiology unit with unstable ischemic cardiopathy. In this context, we review the prognostic value of cardiac biomarkers in CV morbidity and mortality. The particularity of this case was the preemptive assessment of cardiac biomarkers. High serum levels of these biomarkers, in a patient without any cardiac concerns at evaluation moment, should promote for early invasive investigations.

Concomitant invasive pulmonary aspergillosis and Candida zeylanoides bloodstream infection in an acute myeloblastic leukemia patient

Current management of acute myeloblastic leukemia (AML) involves induction chemotherapy followed by risk stratified consolidation approaches, consisting of high dose chemotherapy or allogeneic stem cell transplantation. AML patients are at high risk of invasive fungal disease, particularly mold infections, due to the leukemia and chemotherapy related immune deficit. We present an AML patient with prolonged fever and neutropenia after induction therapy, during which she developed concomitant Candida zeylanoides bloodstream infection and invasive pulmonary aspergillosis. The response to Caspofungin and Voriconazole therapy was delayed, and this dictated the surgical management of the remnant pulmonary lesions. A histological confirmation of aspergillosis was thus obtained. Evaluation of host risk factors for invasive fungal disease, prompt scale-up of the diagnosis scheme and initiation of antifungal therapy are mandatory in order to ensure patient survival.