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Clinical utility of chitotriosidase enzyme activity in nephropathic cystinosis.

Elmonem MA, Makar SH, van den Heuvel L, Abdelaziz H, Abdelrahman SM, Bossuyt X, Janssen MC, Cornelissen EA, Lefeber DJ, Joosten LA, Nabhan MM, Arcolino FO, Hassan FA, Gaide Chevronnay HP, Soliman NA, Levtchenko E - Orphanet J Rare Dis (2014)

Bottom Line: WBC cystine determination forms the basis for the diagnosis and therapeutic monitoring with the cystine depleting drug (cysteamine).Controls with decreased renal function had mild to moderate chitotriosidase elevations; however, their levels were significantly lower than in cystinotic patients with comparable degree of renal insufficiency.In culture, human control macrophages engulfed cystine crystals and released TNF-α into culture supernatant in a crystal concentration dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Chemical Pathology, Inherited Metabolic Disorder Laboratory (IMDL), Cairo University, Cairo, Egypt. mohamed.abdelmonem@kasralainy.edu.eg.

ABSTRACT

Background: Nephropathic cystinosis is an inherited autosomal recessive lysosomal storage disorder characterized by the pathological accumulation and crystallization of cystine inside different cell types. WBC cystine determination forms the basis for the diagnosis and therapeutic monitoring with the cystine depleting drug (cysteamine). The chitotriosidase enzyme is a human chitinase, produced by activated macrophages. Its elevation is documented in several lysosomal storage disorders. Although, about 6% of Caucasians have enzyme deficiency due to homozygosity of 24-bp duplication mutation in the chitotriosidase gene, it is currently established as a screening marker and therapeutic monitor for Gaucher's disease.

Methods: Plasma chitotriosidase activity was measured in 45 cystinotic patients, and compared with 87 healthy controls and 54 renal disease patients with different degrees of renal failure (CKD1-5). Chitotriosidase levels were also correlated with WBC cystine in 32 treated patients. Furthermore, we incubated control human macrophages in-vitro with different concentrations of cystine crystals and monitored the response of tumor necrosis factor-alpha (TNF-α) and chitotriosidase activity. We also compared plasma chitotriosidase activity in cystinotic knocked-out (n = 10) versus wild-type mice (n = 10).

Results: Plasma chitotriosidase activity in cystinotic patients (0-3880, median 163 nmol/ml/h) was significantly elevated compared to healthy controls (0-90, median 18 nmol/ml/h) and to CKD patients (0-321, median 52 nmol/ml/h), P < 0.001 for both groups. Controls with decreased renal function had mild to moderate chitotriosidase elevations; however, their levels were significantly lower than in cystinotic patients with comparable degree of renal insufficiency. Chitotriosidase activity positively correlated with WBC cystine content for patients on cysteamine therapy (r = 0.8), P < 0.001. In culture, human control macrophages engulfed cystine crystals and released TNF-α into culture supernatant in a crystal concentration dependent manner. Chitotriosidase activity was also significantly increased in macrophage supernatant and cell-lysate. Furthermore, chitotriosidase activity was significantly higher in cystinotic knocked-out than in the wild-type mice, P = 0.003.

Conclusions: This study indicates that cystine crystals are potent activators of human macrophages and that chitotriosidase activity is a useful marker for this activation and a promising clinical biomarker and therapeutic monitor for nephropathic cystinosis.

No MeSH data available.


Related in: MedlinePlus

A phase contrast micrograph of macrophages in culture after exposure to cystine crystals. Many macrophages show intracellular crystals (short arrows) and some show pseudopodia (long arrows) to help in movement and engulfment of the crystals.
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Fig3: A phase contrast micrograph of macrophages in culture after exposure to cystine crystals. Many macrophages show intracellular crystals (short arrows) and some show pseudopodia (long arrows) to help in movement and engulfment of the crystals.

Mentions: Uptake of cystine crystals by mature macrophages was demonstrated by phase contrast microscopy (Figure 3). Over 95% of macrophages were still strongly attached to the culture flask surface after 48 hours of crystal elimination denoting cell viability. Looking for direct evidence of macrophage activation after crystal uptake, TNF-α was measured at predetermined intervals. The detected peak level for TNF-α was eight hours after crystal elimination, and the maximum response was obtained with the highest crystal concentration used (200 μg/cm2), which was about ten folds the zero level response (Figure 4).Figure 3


Clinical utility of chitotriosidase enzyme activity in nephropathic cystinosis.

Elmonem MA, Makar SH, van den Heuvel L, Abdelaziz H, Abdelrahman SM, Bossuyt X, Janssen MC, Cornelissen EA, Lefeber DJ, Joosten LA, Nabhan MM, Arcolino FO, Hassan FA, Gaide Chevronnay HP, Soliman NA, Levtchenko E - Orphanet J Rare Dis (2014)

A phase contrast micrograph of macrophages in culture after exposure to cystine crystals. Many macrophages show intracellular crystals (short arrows) and some show pseudopodia (long arrows) to help in movement and engulfment of the crystals.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4269071&req=5

Fig3: A phase contrast micrograph of macrophages in culture after exposure to cystine crystals. Many macrophages show intracellular crystals (short arrows) and some show pseudopodia (long arrows) to help in movement and engulfment of the crystals.
Mentions: Uptake of cystine crystals by mature macrophages was demonstrated by phase contrast microscopy (Figure 3). Over 95% of macrophages were still strongly attached to the culture flask surface after 48 hours of crystal elimination denoting cell viability. Looking for direct evidence of macrophage activation after crystal uptake, TNF-α was measured at predetermined intervals. The detected peak level for TNF-α was eight hours after crystal elimination, and the maximum response was obtained with the highest crystal concentration used (200 μg/cm2), which was about ten folds the zero level response (Figure 4).Figure 3

Bottom Line: WBC cystine determination forms the basis for the diagnosis and therapeutic monitoring with the cystine depleting drug (cysteamine).Controls with decreased renal function had mild to moderate chitotriosidase elevations; however, their levels were significantly lower than in cystinotic patients with comparable degree of renal insufficiency.In culture, human control macrophages engulfed cystine crystals and released TNF-α into culture supernatant in a crystal concentration dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Chemical Pathology, Inherited Metabolic Disorder Laboratory (IMDL), Cairo University, Cairo, Egypt. mohamed.abdelmonem@kasralainy.edu.eg.

ABSTRACT

Background: Nephropathic cystinosis is an inherited autosomal recessive lysosomal storage disorder characterized by the pathological accumulation and crystallization of cystine inside different cell types. WBC cystine determination forms the basis for the diagnosis and therapeutic monitoring with the cystine depleting drug (cysteamine). The chitotriosidase enzyme is a human chitinase, produced by activated macrophages. Its elevation is documented in several lysosomal storage disorders. Although, about 6% of Caucasians have enzyme deficiency due to homozygosity of 24-bp duplication mutation in the chitotriosidase gene, it is currently established as a screening marker and therapeutic monitor for Gaucher's disease.

Methods: Plasma chitotriosidase activity was measured in 45 cystinotic patients, and compared with 87 healthy controls and 54 renal disease patients with different degrees of renal failure (CKD1-5). Chitotriosidase levels were also correlated with WBC cystine in 32 treated patients. Furthermore, we incubated control human macrophages in-vitro with different concentrations of cystine crystals and monitored the response of tumor necrosis factor-alpha (TNF-α) and chitotriosidase activity. We also compared plasma chitotriosidase activity in cystinotic knocked-out (n = 10) versus wild-type mice (n = 10).

Results: Plasma chitotriosidase activity in cystinotic patients (0-3880, median 163 nmol/ml/h) was significantly elevated compared to healthy controls (0-90, median 18 nmol/ml/h) and to CKD patients (0-321, median 52 nmol/ml/h), P < 0.001 for both groups. Controls with decreased renal function had mild to moderate chitotriosidase elevations; however, their levels were significantly lower than in cystinotic patients with comparable degree of renal insufficiency. Chitotriosidase activity positively correlated with WBC cystine content for patients on cysteamine therapy (r = 0.8), P < 0.001. In culture, human control macrophages engulfed cystine crystals and released TNF-α into culture supernatant in a crystal concentration dependent manner. Chitotriosidase activity was also significantly increased in macrophage supernatant and cell-lysate. Furthermore, chitotriosidase activity was significantly higher in cystinotic knocked-out than in the wild-type mice, P = 0.003.

Conclusions: This study indicates that cystine crystals are potent activators of human macrophages and that chitotriosidase activity is a useful marker for this activation and a promising clinical biomarker and therapeutic monitor for nephropathic cystinosis.

No MeSH data available.


Related in: MedlinePlus