Localized Amyloidosis of the Upper Aerodigestive Tract : Complex Analysis of the Cellular Infiltrate and the Amyloid Mass

The aim of this study was to analyse the composition of amyloid mass and the plasmacytic infiltrate of localized amyloidosis of the upper aerodigestive tract.Biopsy materials were studied by light microscopy, immunohistochemistry (IHC), and mRNA in situ hybridization (mRNA-ISH). The amyloid mass was also analysed with high-performance liquid chromatography mass spectrometry- (HPLC-MS-) based proteomics.Nodular and diffuse forms of amyloid deposition were detected. IHC analysis revealed λ-light chain (LC) in two cases, κ-LC in one case. The remaining two were positive with both. Proteins, well known from other amyloidoses like amyloid A (AA), prealbumin/transthyretin (PA), apolipoprotein A-I (ApoAI), and amyloid P component (APC), and also keratin were found with variable intensities in the cases. HPLC-MS revealed dozens of proteins with both LCs in all the lesions but sometimes with surprisingly small intensities. mRNA-ISH analysis revealed identical λ and κ dominance and only one normal κ/λ cell ratio.Cellular infiltrate and protein components in the amyloid showed congruent results in all but one case. The only exception with normal cell ratio and λ-dominant amyloid could be originated from the different protein-secreting activity of plasma cell clones. HPLC-MS analysis explored both LCs in all the amyloid in variable amount, but other proteins with much higher intensities like keratins, apolipoprotein A-IV (ApoAIV), were also detected. Proteins like AA, PA, ApoAI, and APC, previously known about amyloid-forming capability, also appeared. This indicates that localized amyloid in the upper aerodigestive tract is not a homogenous immunoglobulin mass but a mixture of proteins. The sometimes very low light chain intensities might also suggest that not all the localized amyloidosis cases of the upper aerodigestive tract are of convincingly AL type, and the analysis of the cellular infiltrate might indicate that not all are monoclonal

Resiniferatoxin mediated ablation of TRPV1+ neurons removes TRPA1 as well

Resiniferatoxin, the most potent agonist of inflammatory pain/vanilloid receptor/cation channel (TRPV1) can be used for neuron subtype specific ablation of pain generating cells at the level of the peripheral nervous system by Ca(2+)-excytotoxicity. Molecular neurosurgery is an emerging technology either to alleviate severe pain in cancer or treat/prevent different local neuropathies. Our aim was determining sensory modalities that may be lost after resiniferatoxin treatment.status: publishe

Human keratinocytes are vanilloid resistant

BACKGROUND: Use of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca(2+)-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. METHODS: To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. RESULTS: Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca(2+)-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1-50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca(2+)-cytotoxicity ([RTX]>15 microM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca(2+)-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. CONCLUSION: TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials

The TRPV1 protein level in the keratinocytes was under detection limit in Western blotting assay.

<p>Proteins were extracted from human trigeminal ganglion (TG), primary human keratinocytes (NEHK) and the HaCaT cell lines. With a commercially available polyclonal antibody only the TG expressed 94 kDa TRPV1 can be detected (A). The equal loading of protein extracts was validated by β-actin (∼50 kDa), a common, ubiquitously expressed cytoskeletal protein (B).</p

Keratinocytes express low levels of the full length TRPV1 transcript together with transcript of the TRPV1b dominant negative splice form.

<p>Detection of TRPV1 transcript in differentiating human cultured keratinocytes (NHEK) (A) and HaCaT cells (B) by real-time RT-PCR. TRPV1 threshold cycle numbers normalized to the internal control 18S rRNA (delta CT) are plotted. The amount of mRNAs is inversely proportional to the plotted threshold cycle numbers. Data are presented as means of three independent experiments. (*: p<0.05, Dunnet test). The amount of mRNA was also compared in samples from cultured keratinocytes, human HaCaT cells and human trigeminal ganglion (C). The schematic model of TRPV1 and TRPV1b is shown in the corresponing graphs.</p

Transformed cells expressing low levels of TRPV1 were resistant to TRPV1-mediated capsaicin toxicity.

<p>NIH3T3 mouse fibroblasts expressing variable levels of TRPV1-YFP fusion protein were incubated in the absence of capsaicin (A) or with different capsaicin concentrations, 0.1 µM (B), 1 µM (C), 10 µM (D) for 30′, then propidium iodide was added and the flow-cytofluorometric analysis was performed. The M1 and M2 regions contain the intact (propidium iodide negative) TRPV1-YFP high and low positive cells, respectively. Only TRPV1 high positive cells were sensitive to dose dependent, TRPV1 mediated killing by capsaicin (E).</p

Neither endo-, nor exo-vanilloids caused ⁴⁵Ca²⁺-influx in keratinocytes.

<p>Primary human keratinocytes (NHEK), primary culture of DRG neurons, HaCaT keratinocytes and TRPV1 transformed HaCat keratinocytes were seeded in 96-well plates, then incubated for 10-min with different TRPV1 agonists (capsaicin, CAP; resiniferatoxin, RTX; N-arachidonoyl-dopamine, NADA; Anandamide, ANA) and combinations of agonists and antagonists (CAP or RTX+capsazepine, capZ) in the presence of ⁴⁵Ca²⁺. Cell-bound radioactivity was measured with liquid scintillation. The bars represent mean scintillation counts per minute (c.p.m.)+SD of eight parallel samples from a representative experiment repeated three times with similar results.</p

Keratinocytes were not killed by low dose vanilloids.

<p>Primary human keratinocytes (NHEK), proliferating and differentiating HaCaT keratinocytes, TRPV1-expressing HaCaT keratinocytes, HEK293 human embryonal cells, NIH3T3 mouse fibroblasts and sf9 insect cells were grown for 24 hr on 96-well plates in the presence of indicated concentrations of RTX (A) and CAP (B, C). Cell survival was evaluated by the MTS bioassay. Only HaCaT cells stably transformed with TRPV1 transgene were sensitive to cytotoxicity triggered by low dose vanilloids, while normal keratinocytes were only killed by extremely high vanilloid doses, likewise toxic to the negative control insect cells, HEK293 or NIH3T3 cells. Data from a representative experiment repeated three times with similar results is shown. Values are shown as means±SD. (*: p<0.05, t-test).</p

Cobalt histochemistry did not show functional TRPV1 in keratinocytes.

<p>A: TRPV1ε/HaCaT and rat DRG culture (insert) treated with 50 µM RTX in the presence of cobalt cations shows dark CoS precipitate. B: TRPV1ε/HaCaT without RTX C–D: HaCaT and NHEK treated with 50 µM RTX did not produce the characteristic precipitate formed in the presence of intracellular cobalt cations. Bar represents 0.2 mm.</p