Evaluation of plasma chitotriosidase, plasma cytokines and cystine crystal scoring in skin and hair for the non-invasive therapeutic monitoring of nephropathic cystinosis.

General background on cystinosis

Cystinosis is an autosomal recessive disease caused by bi-allelic mutations in
the CTNS gene encoding lysosomal cystine transporter cystinosin [Town et al.
1998]. The disease is characterized by lysosomal cystine tissue accumulation in
all body tissues. Kidneys are first affected with renal Fanconi syndrome being
the initial symptom in the majority of patients. When left untreated renal
disease invariably progresses towards end stage renal failure during the first
decade of life [Gahl et al. 2002]. Extra-renal organs (eyes, endocrine organs,
muscles, gastro-intestinal tract, central and peripheral nervous system) are
also affected by cystinosis, generally later in life [Wilmer et al. 2010]. The
amino thiol cysteamine is the only available drug for decreasing lysosomal
cystine accumulation that delays the progression of renal and extra-renal organ
damage in cystinosis [Markello et al. 1993; Nesterova et al. 2008]. Cysteamine
bitartrate salt is the most widely used cysteamine formulation and is marketed
as an immediate-release preparation (Cystagon* ) that has to be administered
every 6 hours and a delayed-release preparation (Procysbi*) that has to be
administered every 12 hours.
Currently, WBC cystine assay is the gold standard for the diagnosis of
cystinosis and therapeutic monitoring of cysteamine therapy; however, it is
neither ideal nor practical monitoring tool.
It was introduced by Oshima et al. who used [14C] cystine and E.coli
cystine-binding protein [Oshima et al. 1974]. Currently most laboratories
switched to HPLC [de Graaf-Hess et al. 1999] or LC-MS/MS [Chabli et al. 2007]
methods to avoid radioactive exposure. Regardless of the large sample needed to
separate WBC for HPLC or LC-MS/MS cystine assays (*10ml), both methods are
tedious and are not widely available except in very few highly specialized
laboratories worldwide.
While, biochemical cystine determination can be well-standardized, the major
source of imprecision is attributed to WBC isolation and storage [Levtchenko et
al. 2004]. Furthermore, blood for cystine assay must be taken 6 hours after the
last cysteamine dose in case of Cystagon* or 12.5 hours in case of Procysbi*
which is not always clinically feasible.
Cystine preferentially accumulates in polymorphonuclear leucocytes (PMN), thus,
in patients with lymphocyte predominance (due to young age or viral infection);
PMN cells should be used for cystine detection which poses additional technical
problems [Levtchenko et al. 2004]. Furthermore, PMN cells harboring cystine are
very short living cells (*12hours); therefore WBC cystine represents relatively
a short time of therapeutic control. Hypothetically, if a patient complies with
cysteamine treatment strictly few days before the assay, he may appear properly
controlled regardless of previous compliance status.
Therefore, in this research proposal we intend to develop and to evaluate
alternative strategies for monitoring the response to cysteamine therapy in
cystinosis patients.


Macrophage activation by cystine crystals

Recent evidence suggests that the immune system might play a role in the
pathogenesis of nephropathic cystinosis and its rapid progression to ESRD
unlike other types of Fanconi syndromes [Prencipe et al. 2014]. Prencipe et
al. detected the stimulation of inflammasome related cytokines as IL-1β and
IL-18 in human peripheral mononuclear cells when exposed to cystine crystals,
in the plasma of cystinotic patients and in the serum and tissues of Ctns
knocked-out mice.
Chitotriosidase is a fully active chitinase produced by activated macrophages.
Its elevation is documented in several lysosomal storage disorders. [Hollak et
al. 1994, Guo et al. 1995]. Our preliminary data demonstrate that plasma
chitotriosidase activity is significantly elevated in cystinotic patients over
both normal and renal controls. Chitotriosidase activities also correlated with
WBC cystine contents in cysteamine treated patients above 2 years of age.
Control human macrophages were potently activated in-vitro when exposed to
different concentrations of cystine crystals through the significant elevations
of chitotriosidase activity in both supernatant and cell lysate. Furthermore,
chitotriosidase activity was significantly increased in the plasma of
cystinotic knocked-out versus wild-type mice. [Elmonem et al, 2014]

When compared to WBC cystine assay, plasma chitotriosidase is much simpler,
faster, more economic, stable and needs a much smaller sample (1ml or less)
which is more convenient, especially in young children. On the other hand,
chitotriosidase is produced by macrophages which have a very long life-span
(months to years), and therefore should provide a better notion about
therapeutic response over a longer time period.


Confocal microscopy of skin and hair for detection of cystine crystal:

Recently, Chiaverini et al., have shown that reflectance confocal microscopy
(RCM) is able to detect dermal cystine deposition in young patients with
cystinosis. Cystine deposits were visualized as bright, round, or oval-shaped
dermal particles of variable size. These particles appear to be speci*c to
cystinosis, as no particles were identi*ed in control patients. The nature of
the deposits was further confirmed by electron microscopy analysis that showed
that the particles correspond to crystalline cystine deposits within
fibroblasts in the reticular dermis (Chiaverini et al 2013). The examination
was quick (5 minutes), painless, and well tolerated even by the youngest
patients.
RCM scoring of dermal cystine deposition could be also used as a non-invasive
marker of tissue cystine load and hence to cysteamine treatment response.

Based on this background information and our preliminary data in this project
we aim to perform longitudinal study in cystinosis patients to correlate
clinical course under cysteamine therapy and

1) Markers of macrophage activation in patients plasma: chitotriosidase
activity, Interleukin-18 (IL-18) ,interleukin-1beta (IL-1*) and Interleukin-6.

2) Quantitative analysis of cystine crystals in patients* skin and hair by RCM.

This study is has an open label desing.

Fifty cystinotic patients either on cysteamine treatment or at start of
cysteamine treatment will be prospectively recruited and followed up for a
period of 2 years.
Being an orphan disease, patients will be recruited from four different centers
caring for nephropathic cystinosis patients, three in Europe at: UZ Leuven,
Leuven, Belgium; Radboud University Medical Center, Nijmegen, The Netherlands
and Bambino Gesu Pediatric Hospital, Rome, Italy and one in Egypt at: Cairo
University Children Hospital, Cairo, Egypt.

Every 3 months the following parameters will be evaluated:
1- Clinical and basic laboratory evaluation including: serum creatinine,
urinary albumin/creatinine ratio, CRP, corneal cystine crystals (once per year).
2- Cystine assay in WBCs by liquid chromatography tandem mass spectrometry.
Samples will be taken 6 hours after Cystagon* intake or 12.5 hours after
Procysbi* intake.
3- Chitotriosidase activity in plasma by the fluorometric substrate
4-methylumbelliferyl-triacetylchitotrioside.
4- Plasmatic IL-1β and IL-6 levels by IL-1β/IL-1F2 and IL-6 Quantikine HS
ELISA Kit (R&D Systems) and plasmatic IL-18 levels by IL-18 ELISA kit (Medical
and Biologic Laboratories, Nagoya, Japan).
5- Dermal and/or hair cystine crystal score by reflectance confocal microscopy

Not applicable