Compressive Strength of Iliac Bone ECM Is Not Reduced in Osteogenesis Imperfecta and Increases With Mineralization

Osteogenesis imperfecta (OI) is an inheritable, genetic, and collagen-related disorder leading to an increase in bone fragility, but the origin of its “brittle behavior” is unclear. Because of its complex hierarchical structure, bone behaves differently at various length scales. This study aims to compare mechanical properties of human OI bone with healthy control bone at the extracellular matrix (ECM) level and to quantify the influence of the degree of mineralization. Degree of mineralization and mechanical properties were analyzed under dry conditions in 12 fixed and embedded transiliac crest biopsies (control n = 6, OI type I n = 3, OI type IV n = 2, and OI type III n = 1). Mean degree of mineralization was measured by microcomputed tomography at the biopsy level and the mineral-to-matrix ratio was assessed by Raman spectroscopy at the ECM level. Both methods revealed that the degree of mineralization is higher for OI bone compared with healthy control. Micropillar compression is a novel technique for quantifying post-yield properties of bone at the ECM level. Micropillars (d = 5 μm, h = 10 μm) were fabricated using focused ion beam milling and quasi-statically compressed to capture key post-yield properties such as ultimate strength. The qualitative inspection of the stress–strain curves showed that both OI and healthy control bone have a ductile response at the ECM level. The quantitative results showed that compressive strength is not reduced in OI bone and is increasing with OI severity. Nanoindentation measurements revealed that OI bone tends to have a higher Young's modulus, hardness, and dissipated energy compared with healthy bone. Micropillar strength and indentation modulus increased linearly and significantly (p < .0001) with mineral-to-matrix ratio. In conclusion, this study indicates that compressive mechanical properties of dry OI bone at the iliac crest are not inferior to healthy control at the ECM level and increase with mineralization. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Epidemiology and virulence gene expression of intracellular group A streptococci in tonsils of recurrently infected adults

Intracellularly persistent group A streptococci (GAS, Streptococcus pyogenes) have been associated with recurrent tonsillopharyngitis and antibiotic treatment failure. As a supplementation of the published in vitro data, conventional bacteriology and molecular epidemiology was performed on material from 29 adult patients of a German army hospital with anamnestic signs of recurrent tonsillopharyngitis. Pre-surgery tonsil swabs and the surgically removed tonsils were examined with respect to growth of aerobic bacteria in absence and presence of antibiotics with exclusively extracellular activity. Under such antibiotic selection, Staphylococcus aureus and GAS were cultured from specimens of 13 and 3 patients, respectively. In every material GAS-positive by culture methods, the intracellular location of the penicillin-susceptible GAS isolates was confirmed by immunohistologic examination of tonsillar sections using a GAS-specific IgG antibody. The three intracellular GAS isolates were typed by emm gene sequencing and could be associated to types M6 and M49 (two isolates). The bacteria were serially passaged on sheep blood agar, and semiquantitative mRNA analysis from virulence genes was performed using bacteria of the 4th and 25th passage after isolation. An M-type-specific pattern of virulence gene expression and different gene expression levels in relation to the passage number were observed.     

Induction of capsule growth in Cryptococcus neoformans by mammalian serum and CO(2)

The pathogenic fungus Cryptococcus neoformans has a polysaccharide capsule that is essential for virulence in vivo. Capsule size is known to increase during animal infection, and this phenomenon was recently associated with virulence. Although various conditions have been implicated in promoting capsule growth, including CO(2) concentration, osmolarity, and phenotypic switching, it is difficult to reproduce the capsule enlargement effect in the laboratory. In this study, we report that serum can induce capsule growth, and we describe the conditions that induce this effect, not only by serum but also by CO(2). Capsule enlargement was dependent on the medium used, and this determined whether the strain responded to serum or CO(2) efficiently. Serum was most effective in inducing capsule growth under nutrient-limited conditions. There was considerable variability between strains in their response to either serum or CO(2), with some strains requiring both stimuli. Sera from several animal sources were each highly efficient in inducing capsule growth. The cyclic AMP (cAMP) pathway and Ras1 were both necessary for serum-induced capsule growth. The lack of induction in the ras1 mutant was not complemented by exogenous cAMP, indicating that these pathways act in parallel. However, both cAMP and Ras1 were dispensable for inducing a partial capsule growth by CO(2), suggesting that multiple pathways participate in this process. The ability of serum to induce capsule growth suggests a mechanism for the capsular enlargement observed during animal infection.     

Enhancement of peroxisomal enzymes, cytochrome P-452 and DNA synthesis in putative preneoplastic foci of rat liver treated with the peroxisome proliferator nafenopin

The peroxisome proliferator (PP) nafenopin (NAF) enhanced tumordevelopment in rat liver through promotion of a subtype of putativepreneoplastic cell foci, characterized by weak cytoplasmic basophilia(1,2). In order to elucidate the selective growth advantageof these weakly basophilic foci (WBF) we investigated the effectsof NAF on their metabolic phenotype and DNA synthesis. In WBF,as well as in other foci subpopulations and in hepatocellularcarcinomas the occurrence of five NAF-inducible enzymes, i.e.of peroxisomal ß-oxidation (acyl-CoA oxidase, bifunctionalprotein and thiolase), catalase and cytochrome P-452 was studiedby immunohistochemical methods. In untreated livers almost allfoci were stained with the same intensity as the surroundingtissue. When NAF was applied, most of the liver foci showedconsiderably less staining than the non-focal parenchyma inwhich pronounced enzyme induction had occurred. However, thesubpopulation of WBF showed a more heterogeneous pattern ofenzyme expression varying from less to even more than in theadjacent tissue. A similarly broad range of expression of peroxisomalenzymes was found in hepatocellular carcinomas. On average,however, the tumors exhibited less staining and lower activityof peroxisomal ß-oxidation than the surrounding parenchyma.WBF always showed higher rates of DNA synthesis than other focisubtypes and unaltered liver. In     

SHORT COMMUNICATION: Peroxisomal enzyme induction uncoupled from enhanced DNA synthesis in putative preneoplastic liver foci of rats treated with a single dose of the peroxisome proliferator nafenopin

Putative preneoplastic foci of spontaneous origin could be detectedin the livers of 2 year old, untreated male Wistar rats. Theunaltered and preneoplastic hepatocytes showed an identicalexpression of the peroxisomal marker enzyme acyl-CoA oxidase,as determined by immunohistochemical staining. A single doseof the peroxisome proliferator (PP) nafenopin (NAF) inducedthe enzyme predominantly in hepatocytes around the central venulesand cell replication mainly in the periportal areas. However,upon one NAF application almost all of the preneoplastic focishowed a considerably weaker immunoreaction for peroxisomalacyl-CoA oxidase than the surrounding tissue. ConcomitantlyNAF elevated replicative DNA synthesis index in foci up to     

Correlation between antifungal susceptibility testing ofCandida isolates from patients with HIV infection and clinical results after treatment with fluconazole

Summary In an open-label controlled study 23 HIV-infected patients (CDC IV A–E) with documented oropharyngeal candidosis were treated with 100 mg fluconazole orally over 5 days (53 episodes; 1–6 treatments/patient). Efficacy data were compared with a control group of 21 patients who received treatment for 10–21 days with 100 mg fluconazole for candidosis. Candida isolates were repeatedly recovered from patients before and after treatment with fluconazole and antifungal susceptibility testing (microbroth-dilution) was done. Inoculum size, medium pH, incubation time and temperature were standardized. Up to 85% of patients responded to therapy clinically and mycologically.Candida albicans was the most important yeast (86%) isolated from cultures of oral washings. In 90% ofC. albicans isolates MIC to fluconazole were low (1.56 mg/l). Primary resistance to fluconazole was not seen, but secondary resistance occurred in two cases clinically andin vitro (MIC25 mg/l). Short treatment for 5 days was as successful as for 10 to 21 days without leading to significantly more recurrences of oral candidosis in these patients. Selection ofCandida spp. other thanC. albicans (e. g.Candida krusei, Torulopsis glabrata) under repeated fluconazole treatment occurred rarely. One patient developed clinical signs of chronic recurrent candidiasis, where onlyC. krusei could be cultured repeatedly.

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Korrelation zwischen MHK-Bestimmung bei Candida-Isolaten von Patienten mit HIV-Infektion und Therapieverlauf nach Behandlung mit Fluconazol

Zusammenfassung In einer offenen kontrollierten Studie wurde bei 23 HIV-infizierten Patienten (CDC IV A–E) eine dokumentierte oropharyngeale Candidose mit 100 mg Fluconazol oral über 5 Tage behandelt (53 Episoden; 1–6 Episoden/Patient). Die Ergebnisse wurden mit einer Kontrollgruppe verglichen, in der 21 HIV-infizierte Patienten über 10–21 Tage mit 100 mg Fluconazol behandelt wurden. Von den Patienten wurden vor und nach jeder Therapie mit Fluconazol Candida-Isolate gewonnen, differenziert und einer Resistenztestung unterzogen (Mikro-Dilutionstechnik). Für die Resistenztestung wurden Inokulumgröße, pH des Mediums, Inkubationszeit und -temperatur standardisiert. Bis zu 85% der Patienten zeigten klinisch und mykologisch eine Heilung/Besserung.Candida albicans war der am häufigsten isolierte Hefepilz (86%) aus sämtlichen Proben, die mit Mundspülungen gewonnen wurden. 90% allerC. albicans-Isolate warenin vitro Fluconazol-empfindlich (MHK 1,56 mg/l). Eine Primärresistenz gegenüber Fluconazol wurde nicht beobachtet, aber in zwei Fällen trat sowohl klinisch als auchin vitro eine Fluconazol-Resistenz gegenüberC. albicans auf (MHK 25 mg/l). Eine Behandlung der Candidose mit 100 mg Fluconazol über 5 Tage führte zu einer vergleichbaren Heilungs-/Besserungsrate wie über 10–21 Tage, ohne daß die Rezidivrate wesentlich erhöht war. Eine therapiebedingte Selektion von Nicht-albicans-Arten(Candida krusei, Torulopsis glabrata) nach Fluconazol-Behandlung wurde selten beobachtet. Allerdings bestand bei einem Patienten der (klinische) Verdacht auf eine durchC. krusei unterhaltende orale Candidose, da nurC. krusei wiederholt nachgewiesen werden konnte.

     

Response of multicellular tumor spheroids to liposomes containing TNF-α

Summary This publication describes a new model to investigate the influence of tumor necrosis factor- (TNF-) on a three-dimensional glial cell aggregate under defined, standardized, reproducible conditions using the glioma cell line A 172.The cells are initially grown as normal monolayer culture until they reach a cell density of up to 1×10⁶. Subsequently they are grown as spheroids by the liquid overlay technique. Spheroids grown in this way were divided into ten groups of more than 50 cell aggregates. Three groups were coincubated with free TNF- in increasing dosages (100 ng/ml, 200 ng/ml and 1000 ng/ml); three groups were incubated with empty liposomes (0.2 mg/ml, 0.4 mg/ml and 2 mg/ml); three groups received liposomes which had been loaded with TNF-, and one group, which received no treatment, served as control.The diameter of the spheroids ranged from 80 m to 350 m. There was no significant difference in growth between the 3 groups treated with free TNF-. Comparing spheroids treated with TNF- with those which had been coincubated with empty liposomes, there was a significant difference (p<0.001) in growth, which correlated with the amount of liposomes. Similarly, free TNF- had a significantly (P<0.001) stronger growth-inhibiting effect as compared to liposomes loaded with TNF-. Comparing the groups treated with liposomes only to those treated with liposomes loaded with TNF-, the latter exhibited a more marked (although not significantly) growth-inhibiting effect.The preliminary conclusion is that the major growth-inhibiting effect seems to be mediated by the liposomes. This phenomenon is in agreement with results obtained in monolayer cultures.     

An immunohistochemical study of the breast using antibodies to basal and luminal keratins,alpha-smooth muscle actin,vimentin, collagen IV and laminin

Summary The distribution of simple epithelial (K8/18/ 19) and basal (myoepithelial) (K5/14) keratins, -smooth-muscle actin, vimentin, collagen IV and laminin in normal mammary glands and in benign proliferative lesions was studied using monoclonal antibodies (mAbs). These antibodies (Abs) identified myoepithelial cells and luminal cells specifically. In lesions with adenosis and papillomas, the two-layered formation resembled that of normal glands with a purely myoepithelial-epithelial differentiation. In scleradenotic lesions, the main cell was of myoepithelial immunophenotype with intermixed trabecular-tubular proliferations of simple-type epithelium. The sclerosis seems to be the result of an irregular basal lamina synthesis by the myoepithelial cells. In contrast to these lesions, epitheliosis represents a purely intraluminal cell proliferation of clearly simple epithelial immunophenotype and of cells with a basal keratin phenotype, lacking myoepithelial differentiation antigen actin. The basal keratin type epithelium may represent post-stem or intermediate cells developing into luminal epithelium. Epitheliosis appears to be a purely epithelial hyperplasia with striking similarity to the regeneration of normal breast epithelium. The different proliferative patterns may give an explanation for differences in potential cancer risks of patients with these lesions.Dedicated to Prof. Dr. G. Seifert on the occasion of his 70th birthday     

Intestinal non-Hodgkin's lymphoma: a multicenter prospective clinical study from the German Study Group on Intestinal non-Hodgkin's Lymphoma.

PURPOSE: Intestinal non-Hodgkin's lymphomas are not well characterized. We therefore studied prospectively their clinical features and response to standardized therapy. PATIENTS AND METHODS: Fifty-six patients with primary intestinal lymphoma were included in a prospective, nonrandomized multicenter study. Lymphoma resection was recommended and staging was performed according to the Ann Arbor classification. Patients were scheduled to receive six cycles of cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP) chemotherapy, and at stages EIII to EIV, they received additional involved-field radiotherapy. Corticosteroids were used in patients who could not receive chemotherapy. RESULTS: Thirty-five patients had intestinal T-cell lymphoma (ITCL), 21 patients had intestinal B-cell lymphoma (IBCL; 18 diffuse large-cell lymphomas, two marginal-cell lymphomas, and one follicle-center lymphoma). Thirty-four patients at stages EI to EII (14 ITCL and 20 IBCL) and nine patients at stages EIII to EIV (all ITCL) received chemotherapy. No patient in stages EIII to EIV received radiotherapy, because death occurred in 12 of 14 patients. Two-year cumulative survival in patients with IBCL was 94% (95% CI, 82% to 100%) and higher than in patients with ITCL (28% [95% CI, 13% to 43%]; P <.0001), even when only stages EI to EII were considered (ITCL, 37.5% [95% CI, 16.5% to 58.5%]; P <.0001). IBCL patients compared with ITCL patients were at lower lymphoma stages (P <.01), had higher Karnofsky status (P <.005), had intestinal perforation less often (P <.05), required emergency operation less often (P <.05), received CHOP (P <.05) more often, and reached complete remission (P <.0005) more frequently. CONCLUSION: IBCL patients at stages EI and EII respond well to chemotherapy, but the prognosis and treatment of ITCL patients is unsatisfactory.     

Specialized DNA arrays for the differentiation of pancreatic tumors.

PURPOSE: Malignant tumors of the pancreas are frequently indistinguishable from inflammatory tumors arising in the context of a chronic pancreatitis with the use of conventional imaging techniques. Thus, cytologic analysis of cells obtained by abdominal ultrasound, computed tomography, or endoscopic ultrasound-guided fine needle aspiration biopsy is required for diagnosis. However, the reliability of cytologic analyses of pancreatic fine needle aspirates remains unsatisfactory, with a diagnostic accuracy of < or =80%. The purpose of the current study was therefore to develop a novel diagnostic approach based on expression profiling of biopsy material using a specialized diagnostic cDNA array. EXPERIMENTAL DESIGN: Previous gene expression profiling studies were reevaluated to design a 558-feature diagnostic array. Minimal amounts of residual material from pancreatic cytology samples as well as surgically resected tumor and control tissue specimens were analyzed using the diagnostic array and a newly developed statistical classification system. RESULTS AND CONCLUSIONS: Our diagnostic approach resulted in 95% accurate differentiation between ductal adenocarcinomas and nonmalignant tumors of the pancreas. The diagnostic array, in conjunction with conventional diagnostic procedures, is thus suitable to significantly improve the reliability of pancreatic cancer diagnostics and can be expected to become a valuable new tool in the routine workup of suspect masses in the pancreas.