We conduct both basic and clinical investigations on the molecular mechanisms of hereditary neurodegenerative and inflammatory/autoimmune diseases in order to develop novel approaches for the treatment of these diseases. Our studies focus on understanding the regulation and physiological functions of several genes and their products, including uteroglobin (UG), soluble phospholipases A2 (sPLA2s), palmitoyl-protein thioesterase (PPT), and neutral ceramidase. UG is a multifunctional secreted protein with potent sPLA2-inhibitory and immunomodulatory activities. We demonstrated that UG-deficient mice, which we generated, suffer from IgA-nephropathy, the most common primary renal glomerular disease, for which there is no effective treatment. In a related investigation, we study the PPT gene because inactivating mutations in this gene cause infantile neuronal ceroid lipofuscinosis (INCL), a severe neurodegenerative disease of childhood, for which there is no effective treatment. This hereditary disease belongs to a group of common genetic neurodegenerative disorders called Batten disease. Currently, we are conducting a pilot study to determine whether Cystagon™ may be an effective treatment for INCL. We are also using a mouse model of INCL to understand the molecular mechanisms in further detail and to develop novel therapeutic approaches that use gene transfer and embryonic stem (ES) cell technologies.

Role of a Novel Innate Homeostatic Mechan-ism in Counteracting Airway Inflammation and Protecting against Allergic Asthma
Mandal, Zhang, Chowdhury, Ray, Mukherjee; in collaboration with Pattabiraman
In allergic asthma, signalling via the prostaglandin D2 receptor DP mediates airway inflammation by unknown mechanisms. Using three different cell types (i.e., fibroblasts and epithelial and smooth muscle cells) that are constituents of the respiratory system, we demonstrated that DP signalling mediates the activation of nuclear factor (NF)-kB via p38 MAPK, p42/44 MAPK, and PKC pathways in a cell-specific manner, stimulating the expression of COX-2, which is essential for the production of pro-inflammatory PGs. We found that PGD2-induced activation of NF-kB and stimulation of COX-2 expression are drastically inhibited by UG, a low molecular weight, anti-inflammatory/anti-chemotactic protein secreted by the tracheobronchial epithelia of virtually all vertebrates, a most important discovery. Molecular modeling studies indicate that a central hydrophobic cavity created by the UG homodimer sequesters PGD2, abrogating DP signalling (Fig. 13A and B).

Figure 13

A Molecular Model for DP-Signalling and its Regulation by UG
Panel a: Energy-minimized structure of PGD2 docked into the central cavity of the dimer crystal structure of recombinant human UG. The natural form of UG is a homodimer. The human UG dimer structure is represented as ribbons and PGD2 as a van der Waals (space-filling) model. In this figure only the lowest energy structure of the human UG-PGD2 complex is shown. One of the two symmetrically related tyrosines (Y21) in the UG dimer forms a hydrogen bond with the carboxyl group of PGD2 and with the carbon atoms between the two carbon chains, but not with either the hydroxyl oxygen or with the carbonyl oxygen. Panel b: A schematic model of a novel innate mechanism of homeostasis: the molecular mechanism of DP signaling and the effect of UG. We propose that PGD2 binds to the G-protein coupled receptor, DP, and transduces signals in an autocrine as well as paracrine manner. Mediated by p38 MAPK, p42/44MAPK and PKC, PGD2-DP interaction activates NF-kB in a cell-specific manner, which up-regulates the expression of COX-2 that catalyzes the production of proinflammatory prostaglandins from arachidonic acid (AA); cPLA2, whose expression is reported to be stimulated by antigens or allergens and its activation mediated by MAPK, catalyzes the release of AA from cell membrane phospholipids once phosphorylated and activated cPLA2 has been translocated from the cytosol. UG binds and possibly sequesters PGD2, thereby blocking its interaction with DP and preventing the initiation and/or propagation of the inflammatory response.

Finally, using a UGdeficient mouse model of allergic asthma, we demonstrated that pretreatment of these mice with purified recombinant UG (rUG) before allergen challenge prevents COX-2 expression and eosinophil infiltration in the lungs, confirming the results of our in vitro experiments. We propose that DP signalling mediates airway inflammation by NF-kB activation, leading to the expression of one of its target genes, COX-2, which is critical for the production of pro-inflammatory prostaglandins. In this scenario, UG is an essential component of a novel, innate homeostatic mechanism that counteracts inadvertent activation of an allergen-induced inflammatory response in this vital organ and prevents allergic asthma.

Altered Pulmonary Eosinophilic Inflammation in Uteroglobin-Deficient Mice
Zhang, Mukherjee; in collaboration with Huang
The role of UG in modulating pulmonary allergic inflammation was examined in UG-deficient mice generated by targeted gene disruption. Wild-type and homozygous (UG-/-) mice were sensitized with ovalbumin (OVA) and challenged with either OVA or saline. When compared with wild-type mice, OVA-sensitized and challenged UG-/- mice exhibited a significantly higher level of pulmonary eosinophilia as well as significantly increased plasma levels of Th2 cytokines (i.e., IL-4, IL-5, IL-9, and IL-13). In addition, UG-deficient mice exhibited an increased level of eotaxin, but not RANTES, whereas we detected no significant difference in the level of the Th1 cytokine IFN. The results provided the first in vivo evidence that UG plays a role in the modulation of pulmonary allergic inflammation.

Molecular Mechanism of Inhibition of Phospholipase A2 Activity by Uteroglobin
Chowdhury, Zhang, Mukherjee
UG is an anti-inflammatory, secreted protein that inhibits soluble phospholipase A2 (sPLA2) by an unknown mechanism. It is a homodimer, in which each of the 70-amino acid subunits forms four a-helices. We previously reported that the sPLA2-inhibitory activity of UG might reside in a segment of a-helix 3 that is exposed to the solvent. In addition, it has been suggested that UG may inhibit sPLA2 activity by binding and sequestering Ca2+, which is essential for sPLA2 activation. By site-specific mutation, we demonstrated that Lys 43 Glu, Asp 46 Lys, or a combination of the two mutations in the full-length, recombinant human UG (rhUG) abrogates its sPLA2-inhibitory activity. We demonstrated further that recombinant UG does not bind to Ca2+, although when it is expressed with histidine-tag (H-tag), it is capable of binding to Ca2+. Taken together, our results show that Lys 43 and Asp 46 in rhUG are critical residues for the sPLA2-inhibitory activity of UG and that Ca2+-sequestration by rhUG is not likely to be one of the mechanisms responsible for its sPLA2-inhibitory activity.

Clinical Trial of Recombinant Uteroglobin for the Treatment of Neonatal Respiratory Distress Syndrome
Pilon-Clayton, Chowdhury, Zhang, Choi, Mukherjee
Neonatal Respiratory Distress Syndrome (NRDS) is a surfactant deficiency disease that develops in a significant number of premature neonates. Although surfactant replacement therapy is highly successful in resuscitating these neonates, some patients go on to develop a chronic lung disease known as broncho-pulmonary dysplasia (BPD). One potential mechanism of pathogenesis of BPD is degradation of phospholipids in the surfactant, causing the production of pro-inflammatory lipid mediators (e.g., prostaglandins and leukotrienes). Given that UG is naturally present in the newborn and adult lungs but absent from those of premature neonates and is an anti-inflammatory protein, we sought to determine whether intratracheal administration of recombinant human UG (rhUG) is efficacious in treating NRDS and preventing BPD.

The most important milestone in 2002 was, therefore, the completion of this Phase I clinical study of recombinant human uteroglobin (also known as rhCC10) for prevention of bronchopulmonary dysplasia in premature infants with respiratory distress syndrome. In this study, conducted under a collaborative research and development agreement (CRADA) by Claragen Inc., 22 patients were enrolled in three groups: those given a placebo, those given 1.5 mg/kg rhUG, and those given 5 mg/kg rhUG. Despite a higher incidence of necrotizing enterocolitis in the treated groups, the condition was not attributable to the study drug. No issues with safety or tolerability of the rhCC10 were observed. Considerable progress has been made on the GMP manufacturing process for the rhCC10 drug product, increasing yield six-fold by changing the bacterial expression strain as well as by altering the purification procedure to reduce bacterial contaminants and CC10 aggregates. Clinical development of rhCC10 for prevention of neonatal BPD with a Phase II trial as well as further preclinical work for the application of rhCC10 in adult respiratory distress syndrom and asthma will be carried out in the coming year.

Benefit of Cystagon™ for Infant Neuronal Ceroid Lipofuscinoses Patients
Levin, Zhang, Caruso, Gropman, Mukherjee; in collaboration with Fischbeck
Neuronal ceroid lipofuscinoses (NCLs) are the most common (1 in 12,500) heritable progressive encephalopathies of children. Infantile NCL (INCL) is caused by lysosomal PPT deficiency. PPT catalyzes the hydrolysis of thioester linkages in S-acylated polypeptides, and a deficiency causes abnormal accumulation of the polypeptides, leading to INCL. Given that thioester bonds are susceptible to nucleophilic attack, drugs with nucleophilic properties (e.g., Cystagon™ may have therapeutic potential for INCL. Last year, we demonstrated that Cystagon™ not only disrupts thioester linkages in S-acylated polypeptides in cultured cells from INCL patients but also mediates the depletion of intracellular ceroid deposits and prevents their reaccumulation. Taken together, the results suggest that the drug could be an effective treatment for INCL. Given that INCL is a uniformly fatal disease, for which currently there is no effective treatment, and that the active compound of phosphocysteamine has been in clinical use for more than two decades with a proven record of safety, we have implemented a pilot study to determine whether cysteamine bitartrate (Cystagon™) is beneficial for INCL patients. We have received regulatory approval for treating five INCL patients in this study. So far, we have recruited two patients to the protocol, and preliminary evaluations indicate stabilization of retinal functions and of cortical degeneration. The most dramatic results were obtained from the electron microscopic analyses of the white blood cells of these patients. Compared with the cells before treatment, the mononuclear cells show virtually no ceroid depositions in their lysosomes after the initiation of Cystagon™ treatment. So far, the patients do not require any antiepileptic medications. No adverse effects have been encountered with the treatment.

Maintenance of Integrity of Gastrointestinal and Renal Cells by Neutral Ceramidase, Which Prevents Ceramide-Induced Apoptosis
Choi, Anderson, Zhang, Ray, Mukherjee; in collaboration with Popescu
Ceramide, a natural dietary constituent of our daily diet, is a mediator of apoptosis in many cell types. This sphingolipid may also disrupt the mucosal integrity of the gastrointestinal tract by stimulating apoptosis. However, the mechanism that prevents ceramide-induced apoptosis is poorly understood, although it may involve a nonlysosomal ceramidase in the gastrointestinal tract. The enzyme manifests optimal activity in the neutral to alkaline pH (neutral/alkaline ceramidase), degrading ceramide into sphingosine and free fatty acid. In this study, we characterized the murine neutral ceramidase gene, mapped its chromosomal location, determined the tissue-specific expression, and delineated its role in ceramide-induced apoptosis using both in vitro and in vivo systems. Our results show that the murine neutral ceramidase (N-CDase) gene structure is virtually identical to that of the human N-CDase. It is expressed at high levels in mouse kidneys and in specific segments of the upper gastrointestinal tract. We also found that neutral ceramidase mRNA expression is stimulated by its substrate, C2-ceramide. Most important, we found that feeding mice with C2-ceramide or injecting them with a specific inhibitor of neutral ceramidase before feeding ceramide dramatically increased apoptosis in mesangial cells in vitro and in murine gastrointestinal tract in vivo. We propose that neutral ceramidase is part of a novel homeostatic mechanism for preventing apoptosis in vital organs such as the gastrointestinal tract and the kidneys.