Preoperative morphological diagnosis of axillary lymph nodes in a breast center consultation service: evaluation of fine-needle aspiration and core biopsy techniques.

PURPOSE: Preoperative routine examination of axillary lymph nodes (ALN) in breast cancer patients is carried out physically and by ultrasound imaging; unsuspicious nodes will lead to a sentinel node (SN) procedure, suspicious ones require axillary dissection (AD). Pre-operative biopsy techniques like fine needle aspiration (FNA) or core biopsy (CB) may reduce the number of false "suspicious" cases and prevent overtreatment. We evaluated the effectiveness of both biopsy techniques. MATERIALS AND METHODS: After physical and ultrasound examination 241 suspicious ALNs were found in 214 patients. Ultrasound-guided FNA and/or CB procedures were chosen randomly, resulting in 138 FNA and 86 CB. In 17 further events both FNA and CB were employed. The samples were examined in our Cytology lab or in the Pathology Department and the findings correlated with post-operative histological lymph node reports. Patients with histologically proven breast cancer underwent sentinel node biopsy, cytologically or histologically positive FNA/CB-findings prompted ALN dissection. RESULTS: Out of 155 FNA samples 34 were not representative (21.9%), 89 showed no tumor cells (57.4%), 30 showed positive tumor cells (19.4%), leaving two missing. All 103 CB showed representative material, positive in 62 (60.2%) and negative in 41 (39.8%) cases. Correlation with histological reports revealed a statistically non-significant advantage for CB over FNA regarding total accuracy (92.9% vs. 78.3%) and sensitivity (92% vs. 73.7%). CONCLUSIONS: Preoperative CB and alternative FNA are valuable complementary methods of predicting ALN involvement in breast cancer patients and may spare the patient unnecessary ALN dissection.

Phosphoglycerate Kinase-A Novel Streptococcal Factor Involved in Neutrophil Activation and Degranulation.

BACKGROUND: Neutrophils have been proposed as important contributors to the hyperinflammatory responses that are associated with severe invasive Streptococcus pyogenes infections. In particular, streptococcal surface proteins have been implicated as potent neutrophil activators. Here we explore the impact of streptococcus-secreted factors on neutrophil activation and degranulation. METHODS: Primary human neutrophils were exposed to supernatants prepared from cultures of invasive S. pyogenes strains of varying serotypes in the stationary growth phase. Neutrophil activation was assessed by measurement of secreted resistin, an azurophilic granule marker, and by determination of the secretome profile, using mass spectrometry. RESULTS: Marked variation in resistin release and the neutrophil secretome profile were observed following exposure to different strains. A high resistin response was triggered exclusively by SpeB-negative strains, suggesting that at least 1 stimulatory factor is susceptible to SpeB proteolytic degradation. Further analysis, including proteomics and stimulation analyses, identified phosphoglycerate kinase as a stimulatory factor for neutrophils. CONCLUSIONS: Taken together, results of this study reveal a novel secreted streptococcal factor that, in the absence of SpeB, can trigger neutrophil activation and degranulation. This finding is of interest in light of reports of hypervirulent SpeB-negative S. pyogenes variants present during invasive infections.

Differential neutrophil responses to bacterial stimuli: Streptococcal strains are potent inducers of heparin-binding protein and resistin-release.

Neutrophils are critical for the control of bacterial infections, but they may also contribute to disease pathology. Here we explore neutrophil responses, in particular the release of sepsis-associated factors heparin-binding protein (HBP) and resistin in relation to specific bacterial stimuli and sepsis of varying aetiology. Analyses of HBP and resistin in plasma of septic patients revealed elevated levels as compared to non-infected critically ill patients. HBP and resistin correlated significantly in septic patients, with the strongest association seen in group A streptococcal (GAS) cases. In vitro stimulation of human neutrophils revealed that fixed streptococcal strains induced significantly higher release of HBP and resistin, as compared to Staphylococcus aureus or Escherichia coli. Similarly, neutrophils stimulated with the streptococcal M1-protein showed a significant increase in co-localization of HBP and resistin positive granules as well as exocytosis of these factors, as compared to LPS. Using a GAS strain deficient in M1-protein expression had negligible effect on neutrophil activation, while a strain deficient in the stand-alone regulator MsmR was significantly less stimulatory as compared to its wild type strain. Taken together, the findings suggest that the streptococcal activation of neutrophils is multifactorial and involves, but is not limited to, proteins encoded by the FCT-locus.

LL-37 Triggers Formation of Streptococcus pyogenes Extracellular Vesicle-Like Structures with Immune Stimulatory Properties.

Reports have shown that the antimicrobial peptide LL-37 is abundantly expressed but has limited bactericidal effect in Streptococcus pyogenes infections. At sub-inhibitory concentrations, LL-37 has been reported to alter virulence gene expression. Here, we explored the interaction of S. pyogenes strains with LL-37, focusing on bacterial growth, cell surface alterations and pro-inflammatory responses. Bioscreen turbidity measurements of strain 5448 cultured in the presence or absence of LL-37 confirmed the poor antimicrobial effect, and revealed a significant increase in turbidity of bacterial cultures exposed to sub-inhibitory concentrations of LL-37. However, this was not linked to increased bacterial counts. Electron microscopy of LL-37-exposed bacteria revealed the presence of vesicle-like structures on the bacterial surface. The vesicles stained positive for LL-37 and were released from the bacterial surface. Concentrated supernatants enriched in these structures had a broader protein content, including several virulence factors, compared to supernatants from untreated bacteria. The supernatants from LL-37-exposed bacteria were pro-inflammatory and elicited resistin and myeloperoxidase release from neutrophils. This is the first report on S. pyogenes extracellular vesicle-like structures formed at the bacterial surface in response to LL-37. The associated increased pro-inflammatory activity further implicates LL-37 as a potential factor involved in S. pyogenes pathogenesis.

A natural tapasin isoform lacking exon 3 modifies peptide loading complex function.

To assure efficient MHC class I (MHC-I) peptide loading, the peptide loading complex (PLC) recruits the peptide-receptive form of MHC-I, and in this process, tapasin (tpn) connects MHC-I with the peptide transporter TAP and forms a stable disulfide bond with ERp57. Here, we describe an alternatively spliced tpn transcript lacking exon 3, observed in cells infected with human cytomegalovirus. Recognition of exon 3 was regulated via G-runs, suggesting that members of the hnRNP (heterogeneous nuclear ribonucleoprotein)-family regulate expression of the ΔExon3 variant of tpn. Exon 3 includes Cys-95, which is responsible for the disulfide bond formation with ERp57 and, consequently, interaction of the ΔExon3 variant with ERp57 was strongly impaired. Although the ΔExon3 variant specifically stabilized TAP expression but not MHC-I in tpn-deficient cells, in tpn-proficient cells, the ΔExon3 tpn reduced cell surface expression of the tpn-dependent HLA-B*44:02 allele; the stability of the tpn-independent HLA-B*44:05 was not affected. Most importantly, detailed analysis of the PLC revealed a simultaneous binding of the ΔExon3 variant and tpn to TAP, suggesting modification of PLC functions. Indeed, an altered MHC-I ligandome was observed in HeLa cells overexpressing the ΔExon3 variant, highlighting the potential of the alternatively spliced tpn variant to impact CD8(+) T-cell responses.