Immunohistochemistry of thyroid hormones as a functional parameter in thyroid pathology

The authors study by means of immunoperoxidase method the pattern of thyroglobulin, triiodothyronine and thyroxine distribution in 58 cases of thyroid disorders: 15 euthyroid goiters, 10 Graves' disease, 7 Hashimoto's thyroiditis, 11 folliculo-papillary carcinomas (6 primary tumors and 5 lymph node metastases), 8 follicular carcinomas, 4 anaplastic carcinomas and 3 medullary carcinomas. Thyroglobulin, triiodothyronine and thyroxine were present in most of the thyroid disorders, excepting anaplastic and medullary carcinomas. Thyroglobulin and thyroxine were localized both in the follicular epithelium and in the colloid, whereas triiodothyronine was present especially in the follicular cells. The thyroid hormones distribution in benign lesions is rather similar. In carcinomas, the pattern of thyroglobulin, triiodothyronine and thyroxine is more heterogeneous, but generally the triiodothyronine distribution is similar to that of thyroglobulin. In some carcinomas, triiodothyronine and thyroxine showed a weak or negative immunostaining. The immunoperoxidase method is a valuable tool in the study of functional disturbances in the thyroid pathology and in the diagnosis of thyroid carcinoma metastases as well. Positive thyroid hormones staining clearly indicates the thyroid origin of metastases.     

Histopathologic observations on thyroid carcinoma with amyloid stroma.

The authors describe histopathologic aspects in ten cases of thyroid carcinoma with amyloid stroma. This form of cancer represents 3.4% of all the thyroid cancer cases in the last five years in the Institute of Endocrinology. Microscopic examination reveals a polymorphous picture alongside with amyloid deposits. The authors attribute to the tumor cells an important role in producing thyroid amyloidosis.     

Macrophage Specific Caspase-1/11 Deficiency Protects against Cholesterol Crystallization and Hepatic Inflammation in Hyperlipidemic Mice

Background & Aims

While non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis combined with inflammation, the mechanisms triggering hepatic inflammation are unknown. In Ldlr^-/- mice, we have previously shown that lysosomal cholesterol accumulation in Kupffer cells (KCs) correlates with hepatic inflammation and cholesterol crystallization. Previously, cholesterol crystals have been shown to induce the activation of inflammasomes. Inflammasomes are protein complexes that induce the processing and release of pro-inflammatory cytokines IL-1b and IL-18 via caspase-1 activation. Whereas caspase-1 activation is independent of caspase-11 in the canonical pathway of inflammasome activation, caspase-11 was found to trigger caspase-1-dependent IL-1b and IL-18 in response to non-canonical inflammasome activators. So far, it has not been investigated whether inflammasome activation stimulates the formation of cholesterol crystals. We hypothesized that inflammasome activation in KCs stimulates cholesterol crystallization, thereby leading to hepatic inflammation.

Methods

Ldlr ^-/- mice were transplanted (tp) with wild-type (Wt) or caspase-1/11^-/- (dKO) bone marrow and fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks. In vitro, bone marrow derived macrophages (BMDM) from wt or caspase-1/11^-/- mice were incubated with oxLDL for 24h and autophagy was assessed.

Results

In line with our hypothesis, caspase-1/11^-/--tp mice had less severe hepatic inflammation than Wt-tp animals, as evident from liver histology and gene expression analysis in isolated KCs. Mechanistically, KCs from caspase-1/11^-/--tp mice showed less cholesterol crystals, enhanced cholesterol efflux and increased autophagy. In wt BMDM, oxLDL incubation led to disturbed autophagy activity whereas BMDM from caspase-1/11^-/- mice had normal autophagy activity.

Conclusion

Altogether, these data suggest a vicious cycle whereby disturbed autophagy and decreased cholesterol efflux leads to newly formed cholesterol crystals and thereby maintain hepatic inflammation during NASH by further activating the inflammasome.     

Caspase-1 deficiency in mice reduces intestinal triglyceride absorption and hepatic triglyceride secretion

Caspase-1 is known to activate the proinflammatory cytokines IL-1β and IL-18. Additionally, it can cleave other substrates, including proteins involved in metabolism. Recently, we showed that caspase-1 deficiency in mice strongly reduces high-fat diet-induced weight gain, at least partly caused by an increased energy production. Increased feces secretion by caspase-1-deficient mice suggests that lipid malabsorption possibly further reduces adipose tissue mass. In this study we investigated whether caspase-1 plays a role in triglyceride-(TG)-rich lipoprotein metabolism using caspase-1-deficient and wild-type mice. Caspase-1 deficiency reduced the postprandial TG response to an oral lipid load, whereas TG-derived fatty acid (FA) uptake by peripheral tissues was not affected, demonstrated by unaltered kinetics of [³H]TG-labeled very low-density lipoprotein (VLDL)-like emulsion particles. An oral gavage of [³H]TG-containing olive oil revealed that caspase-1 deficiency reduced TG absorption and subsequent uptake of TG-derived FA in liver, muscle, and adipose tissue. Similarly, despite an elevated hepatic TG content, caspase-1 deficiency reduced hepatic VLDL-TG production. Intestinal and hepatic gene expression analysis revealed that caspase-1 deficiency did not affect FA oxidation or FA uptake but rather reduced intracellular FA transport, thereby limiting lipid availability for the assembly and secretion of TG-rich lipoproteins. The current study reveals a novel function for caspase-1, or caspase-1-cleaved substrates, in controlling intestinal TG absorption and hepatic TG secretion.     

Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material

Background

Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.

Method

We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.

Results

Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.

Conclusion

Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.     

The restriction of zoonotic PERV transmission by human APOBEC3G

The human APOBEC3G protein is an innate anti-viral factor that can dominantly inhibit the replication of some endogenous and exogenous retroviruses. The prospects of purposefully harnessing such an anti-viral defense are under investigation. Here, long-term co-culture experiments were used to show that porcine endogenous retrovirus (PERV) transmission from pig to human cells is reduced to nearly undetectable levels by expressing human APOBEC3G in virus-producing pig kidney cells. Inhibition occurred by a deamination-independent mechanism, likely after particle production but before the virus could immortalize by integration into human genomic DNA. PERV inhibition did not require the DNA cytosine deaminase activity of APOBEC3G and, correspondingly, APOBEC3G-attributable hypermutations were not detected. In contrast, over-expression of the sole endogenous APOBEC3 protein of pigs failed to interfere significantly with PERV transmission. Together, these data constitute the first proof-of-principle demonstration that APOBEC3 proteins can be used to fortify the innate anti-viral defenses of cells to prevent the zoonotic transmission of an endogenous retrovirus. These studies suggest that human APOBEC3G-transgenic pigs will provide safer, PERV-less xenotransplantation resources and that analogous cross-species APOBEC3-dependent restriction strategies may be useful for thwarting other endogenous as well as exogenous retrovirus infections.     

Nocturnal oxygen desaturation in patients with congestive heart failure

The aim of our study was to evaluate the modifications of the respiratory pattern during sleeping in patients with congestive heart failure (CHF) by a simple pulse-oxymetry. We studied 10 subjects (8M/2F), mean age 71.4 +/- 12.4 yrs, admitted to sub-intensive cardiological therapy unit, with diagnosis of CHF due to left ventricular insufficiency by ischemic, hypertensive or idiopathic cardiopathy, when in a stable clinical condition. All patients presented arterial blood gas values within normal limits. The ejection fraction of left ventricle showed a mean value of 30.4 +/- 8.2% (range 20%-45%). Nocturnal pulse-oxymetry was performed by pulse-oxymeter (PULSOX 7 Minolta) provided with a digital probe at a sliding speed 24 cm/h. Our data showed that all patients presented nocturnal desaturation episodes (mean oxygen desaturation index 15.7 +/- 18.4). In two patients, we found an "Overlap Syndrome" (obstructive sleep apnoea in presence of cardiopathy). In other patients pulse-oxymetry showed a typical sequence of "fall-rise" basal O2 saturation lasting from 36 to 72 seconds, collected in "wave trains" which were present from 14% to 70% of total sleep time compatible with periodic breathing. In conclusion, our study shows that patients affected by CHF, even if in stable condition and with a PaO2, within normal values, present more or less severe disturbances of nocturnal SaO2, with periodic and regular sequences of SaO2 fall-rise that may be referred to ventilatory troubles such as periodic breathing or Cheyne-Stokes breathing. In these patients the pulse-oxymetry may be considered an efficacious, simple, cheap and well tolerated method.