Metachromatic Granules Research Articles

Sustained Enzymatic Correction by rAAV-Mediated Liver Gene Therapy Protects Against Induced Motor Neuropathy in Acute Porphyria Mice

Acute intermittent porphyria (AIP) is characterized by a hereditary deficiency of hepatic porphobilinogen deaminase (PBGD) activity. Clinical features are acute neurovisceral attacks accompanied by overproduction of porphyrin precursors in the liver. Recurrent life-threatening attacks can be cured only by liver transplantation. We developed recombinant adeno-associated virus (rAAV) vectors expressing human PBGD protein driven by a liver-specific promoter to provide sustained protection against induced attacks in a predictive model for AIP. Phenobarbital injections in AIP mice induced porphyrin precursor accumulation, functional block of nerve conduction, and progressive loss of large-caliber axons in the sciatic nerve. Hepatocyte transduction showed no gender variation after rAAV2/8 injection, while rAAV2/5 showed lower transduction efficiency in females than males. Full protection against induced phenobarbital-attacks was achieved in animals showing over 10% of hepatocytes expressing high amounts of PBGD. More importantly, sustained hepatic expression of hPBGD protected against loss of large-caliber axons in the sciatic nerve and disturbances in nerve conduction velocity as induced by recurrent phenobarbital administrations. These data show for the first time that porphyrin precursors generated in the liver interfere with motor function. rAAV2/5-hPBGD vector can be produced in sufficient quantity for an intended gene therapy trial in patients with recurrent life-threatening porphyria attacks.

Evolution of vacuolar proton pyrophosphatase domains and volutin granules: clues into the early evolutionary origin of the acidocalcisome

BackgroundVolutin granules appear to be universally distributed and are morphologically and chemically identical to acidocalcisomes, which are electron-dense granular organelles rich in calcium and phosphate, whose functions include storage of phosphorus and various metal ions, metabolism of polyphosphate, maintenance of intracellular pH, osmoregulation and calcium homeostasis. Prokaryotes are thought to differ from eukaryotes in that they lack membrane-bounded organelles. However, it has been demonstrated that as in acidocalcisomes, the calcium and polyphosphate-rich intracellular "volutin granules (polyphosphate bodies)" in two bacterial species, Agrobacterium tumefaciens, and Rhodospirillum rubrum, are membrane bound and that the vacuolar proton-translocating pyrophosphatases (V-H+PPases) are present in their surrounding membranes. Volutin granules and acidocalcisomes have been found in organisms as diverse as bacteria and humans.ResultsHere, we show volutin granules also occur in Archaea and are, therefore, present in the three superkingdoms of life (Archaea, Bacteria and Eukarya). Molecular analyses of V-H+PPase pumps, which acidify the acidocalcisome lumen and are diagnostic proteins of the organelle, also reveal the presence of this enzyme in all three superkingdoms suggesting it is ancient and universal. Since V-H+PPase sequences contained limited phylogenetic signal to fully resolve the ancestral nodes of the tree, we investigated the divergence of protein domains in the V-H+PPase molecules. Using Protein family (Pfam) database, we found a domain in the protein, PF03030. The domain is shared by 31 species in Eukarya, 231 in Bacteria, and 17 in Archaea. The universal distribution of the V-H+PPase PF03030 domain, which is associated with the V-H+PPase function, suggests the domain and the enzyme were already present in the Last Universal Common Ancestor (LUCA).ConclusionThe importance of the V-H+PPase function and the evolutionary dynamics of these domains support the early origin of the acidocalcisome organelle. In particular, the universality of volutin granules and presence of a functional V-H+PPase domain in the three superkingdoms of life reveals that the acidocalcisomes may have appeared earlier than the divergence of the superkingdoms. This result is remarkable and highlights the possibility that a high degree of cellular compartmentalization could already have been present in the LUCA.ReviewersThis article was reviewed by Anthony Poole, Lakshminarayan Iyer and Daniel Kahn

Acute Myeloid Leukemia with Mastocytic and Megakaryocytic Differentiation in a Calf

A diagnosis of acute myeloid leukemia was made in a 10-month-old Holstein female calf. The leukemia was macroscopically characterized by great enlargement of the spleen and moderate enlargement of some lymph nodes. Histochemical and immunohistochemical examination disclosed the presence of neoplastic cells either containing metachromatic and tryptase-positive granules or expressing factor VIII-related antigen. The granules, which were positive for naphthol AS-D chloroacetate esterase and did not have particulate contents, were distinct from those of basophilic leukemia cells. This leukemia was thought to be derived from a common myeloid progenitor capable of giving rise to megakaryocyte-erythrocyte progenitors and granulocyte-monocyte progenitors with the ability to differentiate into mast cells.

Pathological study of mycobacterium abscessus keratitis

To characterize the cytopathology, histopathology, immunohistochemistry and ultrastructural organization features of Mycobacterium abscessus keratitis. This retrospective case report included six cases with the lesions tissues scraped from acute phase of mycobacterium abscessus keratitis after corneal foreign body removal. Among the 6 scrapings, 4 were tested for Ziehl-Neelsen acid-fast staining and 3 were examined by transmission electron microscopy (TEM). The corneal tissues of 2 cases in chronic phase of the disease were collected during keratoplasty and were examined by pathological examination including hematoxylin-eosin (HE) staining, acid-fast staining, immunohistostaining and TEM. A single or fascicular red bacilli were observed after acid-fast staining in the four cases of corneal scrapings. The smear samples contained a large number of neutrophils and necrotic corneal epithelium and stroma tissue accompanying with a large number of mycobacteria that were positive for acid-fast staining scattered in and out of inflammatory cells. The corneal buttons obtained at keratoplasty in the 2 patients demonstrated corneal epithelial hyperplasia, stroma degeneration, necrosis and exelcosis, neutrophil and lymphocyte infiltration, neovascularization and fibrous hyperplasia, but no positive mycobacteria by Ziehl-Neelsen acid-fast staining. There were no CD4(+) or CD8(+) cells from immunohistochemistry examination. TEM of the corneal scrapings of the 3 cases showed phagocytosis of mycobacterium abscessus in mononuclear macrophage or neutrophils, inclusion compounds of small vacuoles and metachromatic granules in the thallium. TEM of corneal buttons of 2 cases showed slender rod-shaped mycobacteria with several to dozens of finely ground particles with high electron-dense, but no cell wall (L-form) or inclusion compounds. Mycobacterium abscessus keratitis presents acute purulent inflammatory changes in early stage and advances to chronic and non-specificity inflammation changes in late stage. L-formed mycobacterium abscessus make the disease protraction.

Cutaneous and Tendon Sheath Mastocytomas with Eosinophilic Joint and Tendon Sheath Effusions in a Horse

Cutaneous and Tendon Sheath Mastocytomas with Eosinophilic Joint and Tendon Sheath Effusions in a Horse

Detecting microcalcifications in atherosclerotic plaques by a simple trichromic staining method for epoxy embedded carotid endarterectomies

Atherosclerotic plaques have a high probability of undergoing rapid progression to stenosis, becoming responsible of acute coronary syndrome or stroke. Microcalcifications may act as enhancers of atherosclerotic plaque vulnerability. Considering that calcifications with a diameter smalller than 10 µm in paraffin embedded tissue are rather difficult to detect, our aim was to analyze microcalcifications on semithin sections from epoxy resin embedded samples of carotid endarterectomies using an original trichromic stain (methylene blue-azur B - basic fuchsine - alizarin red). We have compared samples stained either with our method, methylene blue-azur B alone or with Von Kossa staining, and methylene blue-azur B -basic fuchsine alone or with Von Kossa staining.Our method resulted to be simple and fast (ca. 2 min), it gives a sharp general contrast for all structures and allows to easy identify collagen and elastin. In addition, gray-green colour associated to intracellular lipid droplets evidences foam cells, which are particularly abundant in endarterectomies samples. Mast cells and their metachromatic granules are also well recognized. Calcifications over 0,5 µm are clearly recognizable. In conclusion, microcalcifications are clearly distinguished from the extracellular matrix in spite of their reduced dimensions. Methylene blue-azur B-basic fuchsine-alizarin red method is easy to use, reproducible, and is particularly suitable for the identification of microcalcifications in the morphological analysis of atherosclerotic plaques.

La leucodystrophie métachromatique Aspects clinique, biologique et thérapeutique

Scholz's disease or metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a deficiency in arylsulfatase A (ARSA: EC 3.1.6.8). This enzyme is responsible for the degradation of sulfatides commonly called cerebroside-3-sulfate or 3-O-sulfogalactosylcéramide in galactocérébroside and sulfate. The success of hydrolysis of these sphingolipids by ARSA necessarily depends on the presence of saposine B forms a complex with the substrate. The pathological accumulation of sulfatides in the nervous system (myelin, neurons and glial cells) results most often neurological, mental retardation, nervous disorders, blindness. The metachromatic granules accumulated in the central nervous system and peripheral compounds are highly toxic. These are at high levels in the urine of patients affected by the MLD. Arylsulfatase A activity is collapsed in these patients. Unfortunately, the value of enzyme activity is not a predictor of clinical severity of the neuropathology. In contrast, the study of the gene that codes for the ARSA is seen as a way to diagnose the simplest and most reliable of the disease to avoid misdiagnosis due to the presence of pseudodeficit. The conventional therapeutic approaches are essentially symptomatic. They were made in order to restore the enzyme activity of arylsulfatase A and prevent the progression of the pathological accumulation of sulfatides and consequently reduce morbidity associated with MLD.

The Molecular Characterization and Hydrogen Production of a New Species of Anaerobe in Wastewater Treatment Process for H<sub>2</sub> Recovery

The isolation and identification of hydrogen production bacteria(HPB)with high yield and high evolution rate is an important foundation of fermented hydrogen production process by anaerobic digesting high strength organic wastewater. An improved Hungater rolling tubes technique and a plate method of culture bottle (PMCB) were employed to enumerate and isolate the anaerobes. The HPB-RL medium was designed specially for isolating and culturing anaerobic hydrogen bacteria. Culture temperature was at 37°C and 5.5 of pH. About 90 strains of bacteria were isolated, in which, a strain of the hydrogen producer with high yield and high evolution rate was isolated and named Rennanqi12. The isolates were straight rods, Grams positive, strictly anaerobic, no spore-forming bacteria, whole bacteria flagella, having capsule, 3~4 metachromatic granules. The strain of Rennanqi12 could produce hydrogen using molasses wastewater as the carbon resource. The isolate Rennanqi12 has the closest relationship with the strain C. cellulose for the 94% homology of 16S rDNA. The strain of Rennanqi12 might be a new species that belongs to a new genus. The amount of hydrogen-producing (YH2)is 1880.5ml/L medium and the maximum hydrogen-producing rate(QH2)is 28.2 mmol/g•(dry•cell)•h when R12 digested substrates were glucose.

Recent Discoveries on the Roles of Polyphosphates in Plants

Polyphosphate (polyP) has been abundant on Earth long before life began. In living organisms, polyphosphate can be stored in electron-dense granules that have long been referred to as volutin granules. After having being ignored for a long time, this polymer has proven to have numerous unsuspected functions in living organisms and is receiving more attention. The ubiquitous presence of polyP in nature and its structural and physico-chemical characteristics appear to be the drivers for the evolutionary selection of this versatile molecule as a component of cellular processes. Most of our knowledge about the functions of polyP comes from the microbial world. PolyP can influence the transcription and translation of specific genes, act as a metabolic regulator, provide an alternative source of energy, and modulate several stress responses. In plant systems, although the presence of polyP has long been known, its roles in cellular processes have begun to emerge only recently. The purpose of this mini-review is to provide a brief overview of the proposed biological roles of polyphosphates in plants and their roles in microbial/plant interactions.

A simple trichromic staining method detects microcalcifications in atherosclerotic plaques: ex vivo study of human carotid endoarterectomies embedded in epoxy resin

The term vulnerable plaque is used to identify thrombosis-prone plaques and plaques with a high probability of undergoing rapid progression1, thus becoming responsible of acute coronary syndrome or stroke. The role of microcalcifications about 10 μm in diameter in atherosclerotic plaque vulnerability is still debated2-4. From a morphological point of view, microcalcifications are not so easy to detect just by observing paraffin-embedded histopathological sections (3-5 μm thick). Diffuse calcification can be observed using Von Kossa staining5 but such small calcifications cannot be accurately measured. A good characterization of microcalcifications may be achieved observing at light microscopy semithin sections obtained from epoxy resin-embedded tissue samples thinner than 1 μm. Therefore, on the basis of the method reported by D’Amico6 we have developed a simple, rapid and trichromic method, which considers the use of methylene blueazur B, basic fuchsine and alizarin red. Our aim was to analyze microcalcifications on semithin sections from epoxy resin embedded samples of carotid endarterectomies. Comparisons were performed between the present method and methylene blue-azur B (with or without basic fuchsine) followed or not by Von Kossa staining. Our method is simple and fast (ca. 2 min), gives a sharp general contrast for all structures, and allows to easy identify collagen and elastin. In addition, gray-green colour associated to intracellular lipid droplets evidences foam cells, which are particularly abundant in endarterectomies samples. Mast cells and their metachromatic granules are also well recognized. Calcifications over 0,5 μm are clearly recognizable. Microcalcifications are clearly distinguished from the extracellular matrix in spite of their reduced dimensions. Methylene blue-azur B-basic fuchsinealizarin red method is easy to use, reproducible, and is particularly suitable for the identification of microcalcifications in the morphological analysis of atherosclerotic plaques.

Cultivation and Characterization of Canine Skin-Derived Mast Cells

It is essential to develop a technique to culture purified skin-derived mast cells (SMCs) to facilitate immunological research on allergic diseases in dogs. This study was performed to develop an efficient culture system for canine SMCs and to characterize the cells in comparison to canine bone marrow-derived mast cells (BMMCs). Enzymatically digested skin biopsy samples were cultivated in serum-free AIM-V medium supplemented with recombinant canine stem cell factor. Three to five weeks after the initiation of culture, mast cells were collected by a magnetic activated cell separation system using anti-c-Kit antibody. The collected cells were composed of a uniform population showing morphological characteristics of mast cells with a round or oval nucleus and abundant toluidine blue-positive metachromatic granules in the cytoplasm. The results of flow cytometric analysis for the presence of cell membrane c-Kit and Fc epsilon receptor I (FcepsilonRI) indicated that approximately 90% of the cells were mast cells. The cytoplasmic granules were positive for both tryptase and chymase. Apparent dose-dependent degranulation was induced by antibody-mediated cross-linking of immunoglobulin E (IgE) bound to the cells. These cytological and immunological characteristics observed in SMCs were mostly similar to those observed in BMMCs; however, IgE-mediated degranulation was significantly lower in SMCs than BMMCs. The culture system for canine SMCs developed in this study would be useful in understanding the pathophysiology and developing anti-allergic therapeutics in canine allergic dermatitis.

Solitary mastocytoma: tooth eruption as triggering factor

A 2-month-old white male infant presented with a slightly infiltrated, brownish-erythematous plaque on the left arm a few days after birth (following normal physiologic pregnancy and parturition). His parents noted the development of a flaccid bulla on the site of the lesion after a fever and sought the aid of a specialist. The child presented no other general symptoms. At presentation, there was a blood serum-filled flaccid bulla surrounded by seemingly healthy skin (Fig. 1). Rubbing of the skin surrounding the bulla produced a hive-like reaction (Darier's sign). With the suspicion of a cutaneous mastocytoma, an incisional cutaneous punch biopsy was obtained (4 mm in diameter). Figure 1. A blood serum-filled flaccid bulla surrounded by seemingly healthy skin on the left arm of a 2-month-old white male infant Download figure to PowerPoint Histopathologic examination showed a monomorphous and dense proliferation of mast cells within the superficial and deep dermis. Neoplastic cells displayed moderately abundant, oval- or polygonal-shaped cytoplasm and round to oval nuclei (Fig. 2). The nuclei were characterized by clumped chromatin and indistinct or inapparent nucleoli. The cytoplasm was filled with small, faintly visible, eosinophilic or amphiphilic granules which stained metachromatically with Giemsa stain (Fig. 3). Scattered eosinophils were observed throughout the lesion. A final diagnosis of nodular mastocytoma was made. Figure 2. Hematoxylin and eosin stain showing a dermal monomorphous proliferation of cells characterized by moderately abundant, oval- or polygonal-shaped cytoplasm and round to oval nuclei (original magnification, ×20) Download figure to PowerPoint Figure 3. Giemsa stain showing metachromatic small granules within the cell cytoplasm (original magnification, ×20) Download figure to PowerPoint Having followed the lesion's evolution for 3 years, we have observed that it has been affected periodically by blistering, together with sudden episodes of flushing lasting 15–20 min, in the presence of triggering factors such as fevers or vaccinations. The most recent episodes occurred between the age of 15 and 24 months, and were associated with the eruption of deciduous teeth. Over the past few months, with the patient now approximately 3 years of age, the lesion has grown progressively lighter and is beginning to disappear (Fig. 4). Figure 4. After the age of 2 years, the mastocytoma became progressively lighter and smaller Download figure to PowerPoint

Identification of c-kit mutations-independent neoplastic cell proliferation of canine mast cells

Gain-of-function mutations in the proto-oncogene c-kit have been considered the molecular mechanism of neoplastic proliferation of mast cells. However, the importance of c-kit gene mutations is not well evaluated in canine mast cell tumors (MCTs). In the present study, we established and characterized a mast cell line, HRMC, derived from a dog with MCT. We also examined c-kit mutations in HRMC cells and assessed an inhibitory effect of a tyrosine kinase inhibitor, STI571, on HRMC cells. HRMC cells had cytoplasmic metachromatic granules, chymase and tryptase, and expressed both KIT and FcɛRI on the cell surface. HRMC cells contained histamine and released β-hexosaminidase through FcɛRI cross-linking and calcium ionophore stimulation. Nucleotide sequence analysis demonstrated no mutations in an open reading frame of c-kit cDNA and genomic DNA of the juxtamembrane domain of c-kit in HRMC cells. STI571 did not show any inhibitory effects on the proliferation of HRMC cells. These findings clearly demonstrated the existence of c-kit mutations-independent neoplastic canine mast cell proliferation. The growth factor-independent mast cell line established in this study might be valuable to explore novel mechanisms of c-kit mutations-independent neoplastic proliferation of mast cells in dogs.

Immunohistochemical Application of an Antibody Specific for Human CD1a to the Diagnosis of Canine Mast Cell Tumour

Canine mast cell tumours (MCTs) may be graded microscopically for prognostic purposes. Grade I (well-differentiated) and grade II (intermediate differentiation) tumours have an abundance of metachromatic granules within the cytoplasm; however, grade III (poorly differentiated) MCTs may be difficult to diagnose as they frequently have fewer discernable granules. Herein we report that a cross-reactive anti-human CD1a monoclonal antibody (clone O10) may be used in immunohistochemistry to identify canine MCTs of all grades. The antibody was applied to tissue sections from 48 canine MCTs of different histological grades. Serial sections from each tumour were stained with toluidine blue and safranin O to compare diagnostic sensitivity. All MCTs were labelled positively by the CD1a antibody, but histochemical staining was often equivocal and identification of mast cells was extremely difficult in some cases. This antibody did not label neoplastic cells in cases of canine histiocytoma, plasmacytoma or amelanotic melanoma; therefore, the reagent may be a valuable marker for the diagnosis of canine MCTs, especially those tumours of histological grade III.

Skin rash with the histological absence of metachromatic granules as the presenting feature of Hunter syndrome in a 6-year-old boy

Skin rash with the histological absence of metachromatic granules as the presenting feature of Hunter syndrome in a 6-year-old boy

Cytochemistry and morphology of granulocytes of the caecilian Siphonops annulatus (Amphibia, Gymnophiona)

The caecilians (Amphibia, Gymnophiona) constitute one of the least known groups of terrestrial vertebrates because most species live underground in quite inaccessible environments. Siphonops annulatus is an exclusively fossorial species and is the most extensively distributed caecilian in South America. Little is known of this order concerning circulating granulocytes, including their morphological and cytochemical structure and ultrastructure. This paper is part of a project covering the study of granulocytes in representative species of the order Amphibia. Blood extensions were carried out and submitted to Leishman, Toluidine Blue, Periodic acid Schiff, Sirius Red and hydrogen o-toluidine peroxide methods. Part of the samples was prepared for conventional transmission electron microscopy. Among granular leukocytes, mature and immature neutrophils and eosinophils were identified, plus basophils. The most frequent granulocyte encountered in S. annulatus peripheral blood is the neutrophil. This is a cell with a hyper-segmented nucleus and with a very clear cytoplasm when compared to the eosinophil, which presents large cytoplasmic acidophilic granules. On the other hand, the basophils present basophilic and metachromatic granules. Glycogen was detected in the cytoplasm of the neutrophils and eosinophils, while basic protein rich in amino acids was observed in the eosinophil’s granules. Myeloperoxidase activity was detected in the cytoplasm of the neutrophils and eosinophils. Neutrophils were ultrastructurally detected with three types of small granules: eosinophils with large and small spherical granules and basophils with large spherical granules with lamellate structures.

UPTAKE OF PHOSPHORUS BY FILAMENTOUS BACTERIA AND THE ROLE OF CATION ON POLYPHOSPHATES COMPOSITION

ABSTRACT Many microorganisms have the ability to store phosphorus as polyphosphates in volutin granules. The aim of the research was to characterise the phosphorus sequestered by filamentous microorganisms present in the foam. Also the importance of required cations like potassium and magnesium in the process of phosphorus uptake by filamentous microorganisms was examined. Electron microscopy and energy dispersive X – ray analysis were used to define the composition of polyphosphate granules in filamentous bacteria.

The Identification and Characterization of Zebrafish Mast Cells.

Mast cells (MCs), tissue counterparts to mammalian basophils, are known for their role in allergic reactions, inflammation and cancer progression, yet their developmental origin remains controversial due to limitations studying these cells in traditional animal models. The zebrafish provides a highly efficient system for studying vertebrate MC development. All other major hematopoietic lineages have zebrafish counterparts and the fundamental genetic mechanisms controlling hematopoiesis are well conserved. We are the first to identify zebrafish MCs in the gill and intestine. These cells demonstrate classic MC phenotypes including prominent metachromatic granules following staining with toluidine blue and positive immunohistochemical reactions to antibodies against human tryptase and C-KIT. Electron microscopy demonstrates a striking morphologic resemblance to mammalian MCs. Functional studies using the stimulating agent, Compound 48/80 or formalin-killed Aeromonas result in MC degranulation and increased blood levels of key mediators, such as tryptase. These cells also express carboxypeptidase A5 (cpa5), a zebrafish homologue of the human mast cell-specific CPA enzymes. Cpa5 expression in zebrafish embryonic blood cells begins at 24 hours post fertilization and co-localizes with a number of established granulocytic and monocytic markers suggesting that MCs arise from a common granulocyte/monocyte progenitor. Morpholino knockdown studies have demonstrated that the transcription factors gata-2 and pu.1, but not gata-1 are necessary for early MC development. Interestingly, friend of gata-1 (fog-1) may also be required, but in a gata-1 dependent manner. Ongoing morpholino and mutant rescue studies will further establish in vivo the contribution that these transcription factors make to vertebrate MC development. Finally, we have cloned a cpa5 promoter element and shown it can drive expression of the green fluorescent protein in early zebrafish MCs providing a means for generating transgenic zebrafish lines to model human MC diseases for use in high throughput small molecule modifier screens.

Basophilic leukemia in recurrent chronic myelogenous leukemia blast phase

A 31-year-old man with a four-year history of chronic myelogenous leukemia (CML) presents in his second blast phase. The patient’s initial diagnosis was made after routine analysis revealed a white blood cell count (WBC) of 353 10/L. Initial bone marrow biopsy results and cytogenetics were not available for review. The patient responded initially to hydroxyurea followed by imatinib mesylate, at 400 mg daily. A subsequent bone marrow was consistent with morphologic remission; results of cytogenetics and molecular studies were not available. After *3-years, a repeat bone marrow biopsy revealed myeloid blasts comprising 80% of cells consistent with blast phase of CML. Retrospective review of peripheral blood and bone marrow showed only rare (less than 1%) blasts containing granules resembling immature basophils. RT-PCR was positive for BCR-ABL transcript (p210 and p190), and chromosome analysis showed 47, XY, t(2;3)(p23;q29), +8, t(9,22)(q34; q11.2) in 20 of 20 metaphase spreads. Flow cytometric analysis revealed a CD45 positive blast population with moderate side light scatter that strongly expressed CD34, CD117, and CD7 as well as variably expressed CD13, CD33, CD11c, and CD2. Intracellular staining revealed that 1/3 of these cells were myeloperoxidase positive. The imatinib mesylate dose was increased to 800 mg daily but was not tolerated due to nausea. The imatinib mesylate was discontinued, and a second generation ABL kinase inhibitor was initiated. The patient responded to this drug for several months until severe abdominal pain lead to its discontinuation. Less than one week after discontinuation the patient was found to have a WBC of 17 10/L with 30% blasts in peripheral blood, and the tyrosine kinase inhibitor was restarted. Circulating blasts persisted, and the patient underwent induction chemotherapy with an eventual goal of allogeneic transplant. Following this treatment, blasts were no longer detected in peripheral blood, and the patient was thought to be in hematologic remission. After *3-months our patient presented with a WBC of 47 10/L and 76% blasts. These results are consistent with recurrent CML blast phase, though morphologic remission was not established by bone marrow biopsy and persistence of earlier blast crisis remains a possibility. Multiple blast populations were detected morphologically in peripheral blood (Images 1–3), and presence of metachromatic granules after toluidine blue stain indicated a subset (9% of blasts) of basophilic origin (Image 4); maturing basophils were also present (4% of WBC). The patient has since entered a research protocol and had response to a different second generation ABL kinase inhibitor. After 3-weeks of therapy, his WBC has decreased to 1.9 10/L, although occasional blasts (6%) persist. CML is a myeloproliferative disease of bone marrow derived stem cells that contain fusion of BCR on chromosome 22 with c-ABL on chromosome 9 (i.e. Philadelphia chromosome) and which characteristically produces the p210 fusion protein. The initial indolent chronic phase of CML is unstable, and the disease ultimately progresses to accelerated and blast phases. Since the Philadelphia chromosome can be detected in all hematopoietic lineages the blast phase may include neutrophilic, eosinophilic, monocytic, erythroid, megakaryocytic, and basophilic blasts in predominance or in any combination. In about 20–30% of patients the blast phase is due to proliferation of lymphoblasts, and rare patients may have both myeloid and lymphoid blast populations. Acute basophilic leukemia represents <1% of all cases of acute myelogenous leukemia (AML) and must be differentiated from the blast phase of CML, AML with maturation associated with 12p abnormalities or t(6;9), acute eosinophilic leukemia, and acute lymphoblastic leukemia with prominent coarse granules. The persistence of t(9,22), as in the described case, is strong evidence for a predominant basophilic blast population arising from abnormal CML stem cells.

A role for serglycin proteoglycan in granular retention and processing of mast cell secretory granule components

In the absence of serglycin proteoglycans, connective tissue-type mast cells fail to assemble mature metachromatic secretory granules, and this is accompanied by a markedly reduced ability to store neutral proteases. However, the mechanisms behind these phenomena are not known. In this study, we addressed these issues by studying the functionality and morphology of secretory granules as well as the fate of the secretory granule proteases in bone marrow-derived mast cells from serglycin(+/+) and serglycin(-/-) mice. We show that functional secretory vesicles are formed in both the presence and absence of serglycin, but that dense core formation is defective in serglycin(-/-) mast cell granules. The low levels of mast cell proteases present in serglycin(-/-) cells had a granular location, as judged by immunohistochemistry, and were released following exposure to calcium ionophore, indicating that they were correctly targeted into secretory granules even in the absence of serglycin. In the absence of serglycin, the fates of the serglycin-dependent proteases differed, including preferential degradation, exocytosis or defective intracellular processing. In contrast, beta-hexosaminidase storage and release was not dependent on serglycin. Together, these findings indicate that the reduced amounts of neutral proteases in the absence of serglycin is not caused by missorting into the constitutive pathway of secretion, but rather that serglycin may be involved in the retention of the proteases after their entry into secretory vesicles.