The Early Adaptive Immune Response in the Pathophysiological Process of Pneumococcal Meningitis.

BACKGROUND: The adaptive immune system has been considered to play a minimal role in the early host response during bacterial meningitis. METHODS: We investigated the progression and outcome of pneumococcal meningitis in Rag1-/- mice lacking functional B and T cells by assessing overall and symptom-free survival, bacteriological and histological studies, as well as flow cytometry and measurements of proinflammatory mediators. RESULTS: The intracerebral injection of S. pneumoniae D39 induced the recruitment of B and T cells (CD4+, γδ and natural killer) into the brain of wild-type mice. Mice with no functional B and T cells developed clinical symptoms and succumbed to the infection earlier than the wild-type group. In the CNS, Rag1-/- mice showed lower levels of interleukin 1ß, reduced microglial proliferation, and impaired granulocyte recruitment with an earlier spread of pneumococci into the bloodstream, compared with wild-type mice. Lack of B and T cells resulted in a severe impairment of bacterial clearance in blood, spleen, and liver and an exaggerated systemic inflammatory response. CONCLUSIONS: B and T cells are important effector cells delaying the spread of pneumococci from the brain to the systemic circulation and shaping the immune response, thereby prolonging the survival of the host in the absence of antibiotic treatment.

FLT3-ITD and TLR9 use Bruton tyrosine kinase to activate distinct transcriptional programs mediating AML cell survival and proliferation.

Acute myeloid leukemia (AML) is driven by niche-derived and cell-autonomous stimuli. Although many cell-autonomous disease drivers are known, niche-dependent signaling in the context of the genetic disease heterogeneity has been difficult to investigate. Here, we analyzed the role of Bruton tyrosine kinase (BTK) in AML. BTK was frequently expressed, and its inhibition strongly impaired the proliferation and survival of AML cells also in the presence of bone marrow stroma. By interactome analysis, (phospho)proteomics, and transcriptome sequencing, we characterized BTK signaling networks. We show that BTK-dependent signaling is highly context dependent. In Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD)-positive AML, BTK mediates FLT3-ITD-dependent Myc and STAT5 activation, and combined targeting of FLT3-ITD and BTK showed additive effects. In Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD)-negative AML, BTK couples Toll-like receptor 9 (TLR9) activation to nuclear factor κΒ and STAT5. Both BTK-dependent transcriptional programs were relevant for cell cycle progression and apoptosis regulation. Thus, we identify context-dependent oncogenic driver events that may guide subtype-specific treatment strategies and, for the first time, point to a role of TLR9 in AML. Clinical evaluation of BTK inhibitors in AML seems warranted.

Fungal encephalitis in human autopsy cases is associated with extensive neuronal damage but only minimal repair.

AIMS: The present study aimed at examining neuronal injury and repair in post mortem brain sections of humans who died from fungal central nervous system infections. METHODS: Histological and immunohistochemical abnormalities in 15 autopsy cases with fungal central nervous system infections from 1990 to 2008 were compared with findings in 10 age- und sex-matched control cases that died from acute non-neurological causes. The fungal pathogens were identified by culture or polymerase chain reaction and morphology in post mortem tissue. Seven patients with fungal encephalitis had either an organ transplantation or a malignant haematological disorder; five out of 15 did not have a classical predisposing illness but suffered from severe septic infections as the principal cause of immunosuppression, and three from alcoholism. RESULTS: Fungal organisms detected were Aspergillus spp. and other moulds, Candida spp. and black yeast-like fungi including Cladosporium spp. Histological analyses identified microglial activation, astrocytosis and axonal injury in the white matter without additional demyelination as characteristic features of this infectious disease. An increased rate of hippocampal neuronal apoptosis was detected in fungal encephalitis, while the number of recently generated TUC-4 and calretinin-expressing neurones in the dentate gyrus did not differ between patients and controls. CONCLUSIONS: Unlike in other infectious diseases of the nervous system where a coexistence of damage and repair was observed, fungal encephalitis is characterized by strong damage and minimal neuronal regeneration.

Mandibular reconstruction using a calcium phosphate/polyethylene glycol hydrogel carrier with BMP-2.

AIM: To test the hypothesis that a synthetic hydroxyapatite/ß-tricalcium phosphate (HA/TCP) construct combined with polyethylene glycol (PEG) hydrogel including recombinant human bone morphogenetic proteins-2 (rhBMP-2) enhances new bone formation compared with bone morphogenetic proteins-2 (BMP-2) delivered using the HA/TCP construct alone. MATERIAL AND METHODS: Bilateral mandibular partial thickness 20 × 8 × 8 mm (L × W × H) alveolar defects were surgically created in the edentulated posterior mandible in 18 female minipigs. Randomized into two groups of nine animals each, the alveolar defects either received HA/TCP or HA/TCP/PEG with or without BMP-2 (105 µg/defect) in contra-lateral sites using a split-mouth design. Primary outcome, bone density (%) within four regions of interest, was evaluated following a 4-week healing interval when the animals were killed for histometric analysis. RESULTS: Bone morphogenetic proteins-2 loaded onto HA/TCP constructs significantly enhanced new bone formation compared with HA/TCP controls. Adding PEG apparently obstructed BMP-2 induced bone formation. CONCLUSION: Polyethylene glycol compromises the osteogenic effect of BMP-2.

The effect of preoperative hormonal stimulation on the urethral plate; A histologic and histochemical study.

AIM OF THE WORK: Preoperative hormone stimulation (PHS) is used to increase the glans size and may improve the cosmetic appearance after hypospadias surgery. The exact effect of PHS on different penile tissues remains unclear and controversial. Previous studies showed that PHS increased vessel density in the foreskin. However, the effect of PHS on the urethral has never been studied before. In this study we examine the PHS effects on the urethral plate. MATERIALS AND METHODS: Specimens of the urethral plate and the underlying tissue were excised to correct severe chordee in 16 children with proximal and perineal hypospadias with severe chordee in 2 groups. Group A consisted of 8 children received PHS prior to surgery. Group B included 8 children with proximal and perineal hypospadias with severe chordee who did not receive PHS and served as a control group. Specimens were examined blindly using hematoxylin-eosin and elastic van-Gieson stain as well as factor 8 and SMA antibodies. RESULTS: The median age of Group A was 13 (range 10-20) months. The median age of Group B (without prior PHS) was 17 (range 14-29) months (p = 0,03). The median vessel-density in group A (82 vessels per mm2) was significantly higher than in Group B (65 per mm2), (p < 0.05). The median vessel diameter was also significantly higher in Group A (13 µm) than in Group B (11 µm), (p < 0.05). The median epithelial layer thickness in Group A was 110 µm and in Group B 98 µm, but showed no statistical significance (p = 0,16). There was no significant change in dartos fascia layer thickness or androgen receptor expression. There was no visual change in the organization of elastic fibers. CONCLUSION: This study is the first study to document the effect of PHS on the urethral plate in hypospadias with severe chordee. PHS significantly increased the median vessel-density as well as median vessel diameter as compared to a matching control group. The findings of the study may justify PHS administration before the first operation to improve the vascularity and may reduce the severity of chordee.

Hormone receptor expression in hypospadias.

INTRODUCTION: Sex hormone imbalance in utero is hypothesized to play an important role in the pathogenesis of hypospadias. Due to its easy accessibility, foreskin samples have been used to describe hormone receptor expression in rodents, and both adult and pediatric patients. In this study we conducted a systematic approach to assess hormone receptor expression in pediatric patients with hypospadias compared to healthy controls with a focus on age-matching and differences in severity and degree of hypospadias. METHODS: Foreskin samples were collected from 35 children during hypospadias operations (18 distal and 17 proximal hypospadias) and compared with ventral foreskin samples of a control group of 32 children during circumcision (15 age-matched and 17 older boys). The samples were stained with H/E, androgen (AR), estrogen (ER) and progesterone receptors (PR). The receptor stainings were blindly evaluated. An Allred score was used to evaluate receptor expression in both the epithelium as well as stroma. RESULTS: AR was detected in all cases. AR expression in the stroma was more evident than in the epithelium. AR expression in the hypospadias groups was significantly less than the age matched controls (p < 0.05). There was no significant difference between the two hypospadias groups nor between the two control groups. Older control group showed significantly elevated levels of AR expression compared to the hypospadias group (p < 0.05). ER was also detected in all cases. The stroma showed more ER than in epithelium. PR was minimal or negative in all samples. CONCLUSION: Boys with hypospadias showed significantly weaker expression of androgen receptors than age matched controls. The severity of hypospadias did not influence hormone receptor distribution. AR expression is better observed in the stroma than in the epithelium. There was no difference in ER expression between the hypospadias group (distal or proximal) and age matched normal controls. ER was expressed in larger numbers in normal older preputial tissue. The foreskin of prepubertal boys shows little to no expression of PR.

Comparison between cone-beam breast-CT and full-field digital mammography for microcalcification detection depending on breast density.

The purpose of this study was to evaluate the impact of breast density on the diagnostic performance of cone-beam breast-CT (CBBCT) in comparison to full-field digital mammography (FFDM) for the detection of microcalcifications. This retrospective IRB-approved study was conducted between December 2015 and March 2017 and enrolled 171 women with Breast Imaging Reporting and Data System category 4 or 5 lesions on FFDM and additional CBBCT; 56 of which were ineligible. The inclusion was restricted to 83 women (90 breasts, 90 lesions) with microcalcifications. All lesions underwent histology or were monitored by FFDM and a clinical examination at least 2 years after enrollment. Two breast radiologists independently read each data set twice. Sensitivity, specificity and area under the curve were compared between the modalities. Thirty-two breasts (35.5%) were grouped as non-dense breasts (American College of Radiology types a/b) and 58 breasts (64.5%) as dense breasts (American College of Radiology types c/d). Histopathological assessment was performed in 61 of 90 breast lesions (32 malignant, 1 high-risk and 28 benign). Area under the curve was larger for FFDM than for CBBCT (P = .085). The sensitivity was significantly higher for FFDM compared to CBBCT (P = .009). The specificity showed no significant differences comparing FFDM (both readers: 0.62) versus CBBCT (reader 1: 0.76, reader 2: 0.60; P = .192). Inter-observer-reliability on BI-RADS readings was almost perfect for FFDM and moderate for CBBCT (κ = 0.84, κ = 0.54, respectively). Intra-observer agreement was substantial to almost perfect for both methods and readers. Compared with FFDM, CBBCT demonstrated non-comparable results for microcalcification detection in dense and non-dense breasts.

Effects of pulmonary acid aspiration on the lungs and extra-pulmonary organs: a randomized study in pigs.

INTRODUCTION: There is mounting evidence that injury to one organ causes indirect damage to other organ systems with increased morbidity and mortality. The aim of this study was to determine the effects of acid aspiration pneumonitis (AAP) on extrapulmonary organs and to test the hypothesis that these could be due to circulatory depression or hypoxemia. METHODS: Mechanically ventilated anesthetized pigs were randomized to receive intrabronchial instillation of hydrochloric acid (n = 7) or no treatment (n = 7). Hydrochloric acid (0.1 N, pH 1.1, 2.5 ml/kg BW) was instilled into the lungs during the inspiratory phase of ventilation. Hemodynamics, respiratory function and computer tomography (CT) scans of lung and brain were followed over a four-hour period. Tissue samples of lung, heart, liver, kidney and hippocampus were collected at the end of the experiment. RESULTS: Acid instillation caused pulmonary edema, measured as increased extravascular lung water index (ELWI), impaired gas exchange and increased mean pulmonary artery pressure. Gas exchange tended to improve during the course of the study, despite increasing ELWI. In AAP animals compared to controls we found: a) cardiac leukocyte infiltration and necrosis in the conduction system and myocardium; b) lymphocyte infiltration in the liver, spreading from the periportal zone with prominent areas of necrosis; c) renal inflammation with lymphocyte infiltration, edema and necrosis in the proximal and distal tubules; and d) a tendency towards more severe hippocampal damage (P > 0.05). CONCLUSIONS: Acid aspiration pneumonitis induces extrapulmonary organ injury. Circulatory depression and hypoxemia are unlikely causative factors. ELWI is a sensitive bedside parameter of early lung damage.

The urethral plate and the underlying tissue; a histological and histochemical study.

OBJECTIVE: To examine the urethral plate and the underlying tissues in children with proximal hypospadias associated with severe chordee. MATERIALS AND METHODS: The urethral plate and the underlying tissue specimens were excised to correct severe chordee in 17 children with proximal and perineal hypospadias with severe chordee. The median age was 20 months (range 8-36). Sections samples were marked and examined from proximal to distal. Specimens were examined histologically using hematoxylin-eosin (H/E) and Elastic van Gieson (EvG) stain. Histochemical examination was also performed using smooth muscle actin (SMA) and factor 8 antibodies. For control, samples from four patients with hypoplastic urethra proximal to the meatus including the hypoplastic segments until the normal urethra were taken. In addition, the urethra of an adult patient with penile tumor was used as control. RESULTS: The average size of the 17 tissue samples was 0.5 cm × 0.5 cm x 0.3 cm in depth. There was a common pattern that was seen in all the 17 specimens with a variable degree of expression. H/E staining showed that the epithelial lining changed from pseudostratified epithelium at the proximal intact urethra to non-keratinized stratified squamous epithelium at the urethral meatus to keratinized stratified squamous epithelium distally at the urethral plate level. EvG staining showed overall very few elastic fibres that increased slightly in the distal urethral plate. SMA staining showed a circular pattern of smooth muscle cells in the proximal intact urethra that changed to a U-shaped pattern at the level of the meatus, to a triangle shaped pattern just distal to the meatus. The distal urethral plate showed an irregular, disorganized rather flat pattern of the smooth muscles. Factor 8 antibodies staining the blood spaces revealed dysplastic unorganized large blood sinusoids underneath the urethral plate that were different from normal capillaries surrounding the proximal urethra. CONCLUSION: The urethral plate and the underlying tissues in patients with severe chordee have different structure from normal urethra as compared to available literature and the adult control patient. The lack of elastic fibres may help to explain the rigidity of the ventral penis causing chordee. The disorganized irregular distribution of the smooth muscle fibres is suggestive of the hypoplastic corpus spongiosum. The abnormal large blood sinusoids may explain the poor healing quality of the ventral penis in patients with perineal and proximal patients associated with severe chordee. This may explain persistent/recurrent chordee observed later in those patients with severe chordee when dorsal plication is used. The study also supports the recent trend of 2 stage procedure as a plan of management for patients with proximal and perineal hypospadias with severe chordee and excision of all the dysplastic tissues during the first operation.

Loss of inducible nitric oxide synthase expression in the mouse renal cell carcinoma cell line RENCA is mediated by microRNA miR-146a.

Tumor-associated macrophages can potentially kill tumor cells via the high concentrations of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS); however, tumor-associated macrophages actually support tumor growth, as they are skewed toward M2 activation, which is characterized by low amounts of NO production and is proangiogenic. We show that the mouse renal cell carcinoma cell line, RENCA, which, on stimulation, expresses high levels of iNOS mRNA, loses its ability to express the iNOS protein. This effect is mediated by the microRNA miR-146a, as inhibition of RENCA cells with anti-miR- 146a restores iNOS expression and NO production (4.8 ± 0.4 versus 0.3 ± 0.1 µmol/L in uninhibited cells, P < 0.001). In vivo, RENCA tumor cells do not stain for iNOS, while infiltrating tumor-associated macrophages showed intense staining, and both cell types expressed iNOS mRNA. Restoring iNOS protein expression in RENCA cells using anti-miR-146a increases macrophage-induced death of RENCA cells by 73% (P < 0.01) in vitro and prevents tumor growth in vivo. These results suggest that, in addition to NO production by macrophages, tumor cells must produce NO to induce their own deaths, and some tumor cells may use miR-146a to reduce or abolish endogenous NO production to escape macrophage-mediated cell death. Thus, inhibiting miR-146a may render these tumor cells susceptible to therapeutic strategies, such as adoptive transfer of M1-activated macrophages.

Mucoricin is a ricin-like toxin that is critical for the pathogenesis of mucormycosis.

Fungi of the order Mucorales cause mucormycosis, a lethal infection with an incompletely understood pathogenesis. We demonstrate that Mucorales fungi produce a toxin, which plays a central role in virulence. Polyclonal antibodies against this toxin inhibit its ability to damage human cells in vitro and prevent hypovolemic shock, organ necrosis and death in mice with mucormycosis. Inhibition of the toxin in Rhizopus delemar through RNA interference compromises the ability of the fungus to damage host cells and attenuates virulence in mice. This 17 kDa toxin has structural and functional features of the plant toxin ricin, including the ability to inhibit protein synthesis through its N-glycosylase activity, the existence of a motif that mediates vascular leak and a lectin sequence. Antibodies against the toxin inhibit R. delemar- or toxin-mediated vascular permeability in vitro and cross react with ricin. A monoclonal anti-ricin B chain antibody binds to the toxin and also inhibits its ability to cause vascular permeability. Therefore, we propose the name 'mucoricin' for this toxin. Not only is mucoricin important in the pathogenesis of mucormycosis but our data suggest that a ricin-like toxin is produced by organisms beyond the plant and bacterial kingdoms. Importantly, mucoricin should be a promising therapeutic target.

Tumor-associated macrophages in clear cell renal cell carcinoma express both gastrin-releasing peptide and its receptor: a possible modulatory role of immune effectors cells.

PURPOSE: Renal cell carcinomas (RCC) frequently express the gastrin-releasing peptide receptor (GRP-R). Gastrin-releasing peptide (GRP) stimulates tumor cell proliferation and neoangiogenesis. Tumor-associated macrophages (TAM) comprise an important cellular component of these tumors. We analyzed the GRP/GRP-R network in clear cell RCC (ccRCC) and non-clear cell RCC (non-ccRCC) with special regard to its expression by macrophages, tumor cells and microvessels. METHODS: Gastrin-releasing peptide and GRP-R expression in 17 ccRCC and 9 non-ccRCC were analyzed by RT-PCR, immunohistochemistry and double immunofluorescence staining. RESULTS: Tumor-associated macrophages expressed GRP and GRP receptor in ccRCC. Tumor cells and microvessels showed low to intermediate GRP-R expression in nearly all cases. In 12 ccRCC tumor epithelia also expressed low levels of GRP. Microvascular GRP expression was found in nine cases of ccRCC. For non-RCC, the expression of GRP and GRP receptor expression pattern was similar. CONCLUSIONS: Tumor-associated macrophages are the main source of GRP in RCC. GRP receptor on TAM, tumor epithelia and microvessels might be a molecular base of a GRP/GRP receptor network, potentially acting as a paracrine/autocrine modulator of TAM recruitment, tumor growth and neoangiogenesis.

Of Cestodes and Men: Surgical Treatment of a Spinal Hydatid Cyst.

BACKGROUND: The cestode Echinococcus granulosus causes hydatid disease. In addition to manifestations in the liver and lung, it can lead to cystic lesions in the spine. CASE DESCRIPTION: We report a 42-year-old male patient with primary hydatid disease in the eighth thoracic vertebra. The only clinical symptom was chronic back pain. Although laboratory findings were normal, imaging displayed lytic destruction that raised the suspicion of a metastatic disease. Diagnostics of the thoraces and abdomen did not reveal other pathologic abnormalities. Follow-up magnetic resonance imaging (MRI) depicted a progressive compression of the spinal cord and inhomogeneous structure in the fat-suppressed sequences. Because the Jamshidi biopsy was inconclusive, the tumor board recommended surgery. Dorsal decompression, spondylodesis of T6-T10, and vertebral column resection of T8 with complete cyst removal were performed. The resected vertebrae showed a mucous-like lesion with white granular tissue interfusing the whole vertebral body. A pathologic examination and enzyme-linked immunosorbent assay confirmed E. granulosus. Thus chemotherapy with albendazole was initiated. A follow-up MRI of the whole spine confirmed complete remission and found no additional resettlements. The patient's back pain was resolved without neurologic deficits. CONCLUSIONS: For lytic manifestations of the vertebral column, hydatid cysts should be considered a differential diagnosis in addition to malignant metastasis, tuberculosis, and osteomyelitis. Thorough surgical resection and strict follow-up are necessary.

Breast lesion size assessment in mastectomy specimens: Correlation of cone-beam breast-CT, digital breast tomosynthesis and full-field digital mammography with histopathology.

To compare the accuracy of breast lesion size measurement of cone-beam breast-CT (CBBCT), digital breast tomosynthesis (DBT) and full-field digital mammography (FFDM).Patients scheduled for mastectomy due to at least 1 malignant breast lesion were included. Mastectomy specimens were examined by CBBCT, DBT, FFDM, and histopathology.A total of 94 lesions (40 patients) were included. Histopathological analyses revealed 47 malignant, 6 high-risk, and 41 benign lesions. Mean histopathological lesion size was 20.8 mm (range 2-100). Mean absolute size deviation from histopathology was largest for FFDM (5.3 ±â€Š6.7 mm) and smallest for CBBCT 50 mA, high-resolution mode (4.3 ±â€Š6.7 mm). Differences between imaging modalities did not reach statistical significance (P = .85).All imaging methods tend to overestimate breast lesion size compared to histopathological gold standard. No significant differences were found regarding size measurements, although in tendency CBBCT showed better lesion detection and cT classification over FFDM.

Generation of highly differentiated BHY oral squamous cell carcinoma multicellular spheroids.

Three-dimensional (3D) multicellular spheroids (MCS) are considered suitable models in cancer research and anticancer drug development. Although studying the complex tumour characteristics from all different degrees of malignancy is vital, MCS generation has only been described in a few moderately- and poorly differentiated oral squamous cell carcinoma (OSCC) cell lines. No previous study has demonstrated the MCS formation in a highly differentiated OSCC cell line. For the first time, the present study aimed to generate MCS from the highly differentiated OSCC cell line BHY. BHY spheroids were grown in three independent experiments in 96-well plates through the use of the liquid overlay technique. Although BHY cells are grow slowly and are difficult to culture, they formed compact MCS within 24 h. After 3 days of incubation, no further increase in spheroid size was observed. MCS were harvested, paraffin-embedded and 2 µm tissue sections were prepared for further analysis. The diameter and volume of each spheroid were determined. BHY MCS diameter ranged between 46.76 and 233.26 µm, with a volume range from 5.35×104-6.65×106 µm3. In conclusion, using the liquid overlay technique, the highly differentiated OSCC cell line BHY forms different sized spheroids, which may be used for further investigations.

Establishment and characterization of two distinct malignant mesothelioma cell lines with common clonal origin.

We describe two newly established malignant mesothelioma (MM) cell lines derived from a pleural effusion of a male. One cell line, designated as MM-Z03E, reveals an epithelioid cobblestone morphology, while the second one, designated as MM-Z03S and subcloned after in vivo selection, exhibits a sarcomatoid storiform growth pattern. Both cell lines showed the immunologic profile characteristic for MM (i.e., expression of cytokeratin, CK18, calretinin, and vimentin in both phenotypes). Cytogenetics, multicolor fluorescence in situ hybridization, comparative genomic hybridization, and oligonucleotide array CGH were performed on both cell lines. Aberrations shared by both cell lines included chromosomal losses of 1q34 approximately qter, 4, 9p, 10p, 13, 14, 16q, 18, and 22, as well as a complex structural aberration involving chromosome 17. Aberrations exclusive to MM-Z03E included gains of 3q11q27 and 5p, while gain of 9q and losses of 3q27qter, 11q, and 18 in MM-Z03S were exclusive to MM-Z03E. Both cell lines were able to develop solid transplant tumors in nude mice within 16 weeks, and immunophenotyping of tumor xenografts revealed an overall retained expression profile of the markers used. Remarkably, one xenograft from MM-Z03E revealed overexpression of p53 and widely invasive growth. In conclusion, both cell lines are useful in vivo and in vitro model systems to study the underlying genetic mechanisms of biphasic differentiation in MM, which can be of certain value considering the increasing relevance of assessing MM tumor biology for the clinical management of this disease.