Cell surface adenylate kinase activity regulates the F(1)-ATPase/P2Y (13)-mediated HDL endocytosis pathway on human hepatocytes.

We have previously demonstrated on human hepatocytes that apolipoprotein A-I binding to an ecto-F(1)-ATPase stimulates the production of extracellular ADP that activates a P2Y(13)-mediated high-density lipoprotein (HDL) endocytosis pathway. Therefore, we investigated the mechanisms controlling the extracellular ATP/ADP level in hepatic cell lines and primary cultures to determine their impact on HDL endocytosis. Here we show that addition of ADP to the cell culture medium induced extracellular ATP production that was due to adenylate kinase [see text] and nucleoside diphosphokinase [see text] activities, but not to ATP synthase activity. We further observed that in vitro modulation of both ecto-NDPK and AK activities could regulate the ADP-dependent HDL endocytosis. But interestingly, only AK appeared to naturally participate in the pathway by consuming the ADP generated by the ecto-F(1)-ATPase. Thus controlling the extracellular ADP level is a potential target for reverse cholesterol transport regulation.

The nucleotide receptor P2Y13 is a key regulator of hepatic high-density lipoprotein (HDL) endocytosis.

Cell surface receptors for high-density lipoprotein (HDL) on hepatocytes are major partners in the regulation of cholesterol homeostasis. We recently identified a cell surface ATP synthase as a high-affinity receptor for HDL apolipoprotein A-I (apoA-I) on human hepatocytes. Stimulation of this ectopic ATP synthase by apoA-I triggered a low-affinity-receptor-dependent HDL endocytosis by a mechanism strictly related to the generation of ADP. This suggests that nucleotide G-protein-coupled receptors of the P2Y family are molecular components in this pathway. Only P2Y1 and P2Y13 are present on the membrane of hepatocytes. Using both a pharmacological approach and small interference RNA, we identified P2Y13 as the main partner in hepatic HDL endocytosis, in cultured cells as well as in situ in perfused mouse livers. We also found a new important action of the antithrombotic agent AR-C69931MX as a strong activator of P2Y13-mediated HDL endocytosis.

Association of end-product adducts with increased IgE binding of roasted peanuts.

Recently, we have shown that roasted peanuts have a higher level of IgE binding (i.e., potentially more allergenic) than raw peanuts. We hypothesized that this increase in IgE binding of roasted peanuts is due to an increased levels of protein-bound end products or adducts such as advanced glycation end products (AGE), N-(carboxymethyl)lysine (CML), malondialdehyde (MDA), and 4-hydroxynonenal (HNE). To support our hypothesis, we produced polyclonal antibodies (IgG) to each of these adducts, determined their levels in raw and roasted peanuts, and examined their ability to bind to IgE from a pooled serum of patients with clinically important peanut allergy. Results showed that AGE, CML, MDA, and HNE adducts were all present in raw and roasted peanuts. Roasted peanuts exhibited a higher level of AGE and MDA adducts than raw peanuts. IgE was partially inhibited in a competitive ELISA by antibodies to AGE but not by antibodies to CML, MDA, or HNE. This indicates that IgE has an affinity for peanut AGE adducts. Roasted peanuts exhibited a higher level of IgE binding, which was correlated with a higher level of AGE adducts. We concluded that there is an association between AGE adducts and increased IgE binding (i.e., allergenicity) of roasted peanuts.

Feline leukemia virus detection in corneal tissues of cats by polymerase chain reaction and immunohistochemistry.

OBJECTIVES: To determine the presence of feline leukemia virus (FeLV) in the corneas of FeLV-infected cats. ANIMALS STUDIED: Thirty-four random source cats. PROCEDURES: Seventeen cats were found positive for FeLV serum p27 antigen by enzyme-linked immunosorbent assay (ELISA). Twelve ELISA positive cats were also positive on peripheral blood samples by immunofluorescent antibody (IFA) testing. Seventeen ELISA negative cats served as negative controls. Full thickness corneal specimens were collected from the left cornea of all cats and analyzed for FeLV proviral DNA and gp70 antigen by polymerase chain reaction (PCR) and immunohistochemical (IHC) testing, respectively. RESULTS: Eleven (64.7%) positive corneal PCR results were obtained from 17 ELISA positive cats. Of 12 cats that were both ELISA and IFA positive on peripheral blood, 10 (83.3%) had positive corneal PCR results. All corneal tissues from ELISA negative cats were PCR negative. IHC staining of corneal sections revealed the presence of FeLV gp70 in corneal tissues of nine (52.9%) ELISA positive cats. Of the 12 cats that were both ELISA and IFA positive on peripheral blood, eight (66.7%) had positive corneal IHC results. Positive IHC staining was localized to the corneal epithelium. Corneal tissues of all ELISA negative cats and all IFA negative cats were negative on IHC testing. CONCLUSIONS: FeLV antigens and proviral DNA are present in corneal tissues of some FeLV-infected cats. Screening corneal donors for FeLV infection is warranted.

Pyrogenicity and cytokine-inducing properties of Streptococcus pyogenes superantigens: comparative study of streptococcal mitogenic exotoxin Z and pyrogenic exotoxin A.

Streptococcal mitogenic exotoxin Z (SMEZ), a superantigen derived from Streptococcus pyogenes, provoked expansion of human lymphocytes expressing the Vbeta 2, 4, 7 and 8 motifs of T-cell receptor. SMEZ was pyrogenic in rabbits and stimulated the expression of the T-cell activation markers CD69 and cutaneous lymphocyte-associated antigen. A variety of cytokines was released by human mononuclear leukocytes stimulated with SMEZ, which was 10-fold more active than streptococcal pyrogenic exotoxin A. Th2-derived cytokines were elicited only by superantigens and not by streptococcal cells.

Ocular pharmacology.

Many new drugs come on the market each year, and some very useful ones become unavailable. Medical treatment of some of the more common ophthalmic conditions observed in small animal practice are discussed in this article. Selection of the appropriate antibiotic or antiviral is critical for effective treatment, and indications for these drugs are provided. Anti-inflammatory medications and their indications and contraindications are also discussed. Medical therapy of glaucoma is also addressed.

Intraosseous approach to the nasolacrimal duct for removal of a foreign body in a dog.

A Labrador retriever was evaluated because of chronic mucopurulent discharge from the left eye. A foreign body was identified in the nasolacrimal duct by use of dacryocystorhinography. Attempts to alleviate the inflammation by use of flushing and administration of antimicrobials were unsuccessful. At surgery, the infraorbital foramen was used as a landmark for a skin incision, because the nasolacrimal duct courses dorsal and parallel to the infraorbital canal. An air drill was used to remove the portion of the maxillary bone overlying the nasolacrimal duct, which exposed the intraosseous portion of the duct and allowed removal of a plant-material foreign body. The incision in the duct was allowed to heal by second intention, and the dog recovered without complications.

The effects of roasting on the allergenic properties of peanut proteins.

BACKGROUND: Because of the widespread use of peanut products, peanut allergenicity is a major health concern in the United States. The effect or effects of thermal processing (roasting) on the allergenic properties of peanut proteins have rarely been addressed. OBJECTIVE: We sought to assess the biochemical effects of roasting on the allergenic properties of peanut proteins. METHODS: Competitive inhibition ELISA was used to compare the IgE-binding properties of roasted and raw peanut extracts. A well-characterized in vitro model was used to test whether the Maillard reaction contributes to the allergenic properties of peanut proteins. The allergic properties were measured by using ELISA, digestion by gastric secretions, and stability of the proteins to heat and degradation. RESULTS: Here we report that roasted peanuts from two different sources bound IgE from patients with peanut allergy at approximately 90-fold higher levels than the raw peanuts from the same peanut cultivars. The purified major allergens Ara h 1 and Ara h 2 were subjected to the Maillard reaction in vitro and compared with corresponding unreacted samples for allergenic properties. Ara h 1 and Ara h 2 bound higher levels of IgE and were more resistant to heat and digestion by gastrointestinal enzymes once they had undergone the Maillard reaction. CONCLUSIONS: The data presented here indicate that thermal processing may play an important role in enhancing the allergenic properties of peanuts and that the protein modifications made by the Maillard reaction contribute to this effect.

Evaluation of aqueous tear production in dogs following general anesthesia.

Pre- and postanesthetic Schirmer tear test (STT) values were measured in 46 dogs. All subjects had normal preanesthetic STT values (18.3 +/- 2.8 mm per min in the left eye [OS] and 18.3 +/- 3.0 mm per min in the right eye [OD]). Significant differences were found between pre- and postanesthetic STT values. Significant decreases in tear production were evident for up to 24 hours following the anesthetic event. Subject age did not significantly influence the results. Duration of anesthesia significantly affected the rate of return to preanesthetic STT values, with anesthetic events greater than two hours in duration having a prolonged effect as compared to anesthetic events less than two hours in duration. Anticholinergic administration prior to or during anesthesia further lowered postanesthetic STT values.

Equine conjunctival pseudotumors.

Five horses presented with unilateral pink, smooth, nonulcerated conjunctival masses with histologic features characteristic of inflammatory pseudotumors, i.e. proliferative inflammatory lesions clinically resembling true neoplasia. Although causes for the inflammatory lesions were not determined, based on the presence histologically of mononuclear (predominantly lymphocytic) inflammatory cell infiltrates and the absence of infectious agents, parasites or foreign bodies, an immune-mediated pathogenesis was suspected. Affected horses ranged from 5 to 8 years of age with no apparent breed or sex predilection. Conjunctival lesions were nodular in two cases and relatively flat and more diffuse in three cases. Third eyelid lesions were present in three cases and two affected eyes had corneal involvement. Based on findings from these five cases, the prognosis for equine conjunctival pseudotumors appears to be good when lesions are treated by partial or complete surgical excision, local administration of anti-inflammatory agents, or a combination of surgery and anti-inflammatory therapy.

Effect of topical 1% atropine sulfate on intraocular pressure in normal horses.

OBJECTIVE: To determine the effect of topical 1% ophthalmic atropine sulfate on intraocular pressure (IOP) in ocular normotensive horses. Animals Studied Eleven clinically healthy horses. Procedures IOP was measured bilaterally twice daily, at 8 AM and 4 PM, for 5 days. No medication was applied for the first 2 days of the study. Thereafter, one eye of each horse was treated with 0.1 mL of topical 1% atropine sulfate ointment twice daily (7 AM and 7 PM) for 3 days. The contralateral eye served as a control. In eight of the horses, an additional IOP reading was taken 3 days following cessation of the atropine treatment. RESULTS: There was no significant difference in the IOP of control vs. treatment eyes in the pretreatment period, days 1 and 2 (P = 0.97 and 0.55, respectively). During the treatment period, treated eyes of 10 of the horses had significantly lower IOP than control eyes (P = 0.03). The mean IOP reduction in treated eyes, relative to untreated eyes, was 11.2%. One horse had a significant rise in IOP in the treated eye compared to the remaining study animals. The IOP of control eyes did not vary significantly over the observation period (P = 0.27). There was no significant variation in IOP between the 8 AM and 4 PM measurement (P = 0.9). CONCLUSIONS: Topical 1% atropine sulfate causes a small, but significant decline in IOP in most ocular normotensive horses. Because topical atropine may elevate IOP in some horses, it should be used with caution in the treatment of glaucoma in this species.

Inferomedial placement of a single-entry subpalpebral lavage tube for treatment of equine eye disease.

The objective of this study was to describe method of placement, and frequency and severity of complications associated with a subpalpebral lavage system placed in the medial aspect of the equine inferior eyelid. The inferomedial subpalpebral lavage (ISPL) tube is positioned deep in the medial aspect of the inferior conjunctival fornix so that the footplate lies flat between the lower eyelid and the anterior surface of the nictitans. Retrospective data from the placement of 92 ISPL systems placed in 86 horses during a 31-month period were examined. Tube placement was performed using sedation and regional anesthesia only in 59% of horses. The median duration of tube placement was 19 days (range: 1-61 days). Seventy-one horses were treated for up to 55 days following discharge from hospital with an ISPL tube in place. No complications were reported with 59% of ISPL systems. Non-ocular complications were found in 38% of ISPL systems and included tube displacement from the conjunctival fornix (18%), suture loss requiring resuturing of the system to the horse's head (14%), and damage necessitating replacement of the injection port (6%). Ocular complications were recorded in 3% of horses and were limited to inferior eyelid swelling. Vision was retained in 88% of horses. The ISPL system is easily and safely placed, and well tolerated for extended periods. It appears to be associated with infrequent and minor complications when compared with placement of subpalpebral lavage tubes in the superior eyelid.

Allergenicity of Maillard reaction products from peanut proteins.

It is known that peanut allergy is caused by peanut proteins. However, little is known about the impact of roasting on the allergenicity of peanuts. During roasting, proteins react with sugars to form Maillard reaction products, which could affect allergenicity. To determine if the Maillard reaction could convert a nonallergenic peanut protein into a potentially allergenic product, nonallergenic lectin was reacted with glucose or fructose at 50 degrees C for 28 days. Browning products from heat-treated peanuts were also examined. The products were analyzed in immunoblot and competitive assays, using a pooled serum (i.e., IgE antibodies) from patients with peanut anaphylaxis. Results showed that the products were recognized by IgE and had an inhibitory effect on IgE binding to a peanut allergen. Thus, the findings suggest that these Maillard reaction products are potentially allergenic and indicate the need to verify whether the Maillard reaction products formed in peanuts during roasting increase their allergenicity.

Acquisition of a stimulatory activity for Vgamma9/Vdelta2 T cells by a Burkitt's lymphoma cell line without loss of HLA class I expression.

Daudi Burkitt's lymphoma cells activate Vgamma9/Vdelta2 T cells through TCR ligation by an unknown antigen. This activity is for a large part revealed by their lack of HLA class I antigen expression, allowing their escape from KIR downregulation. We characterize here a culture variant of the Burkitt's lymphoma line Raji, RJ-A3, which is able to promote as efficiently as Daudi cells the outgrowth of Vgamma9/Vdelta2 T cells in cocultures in spite of unchanged HLA class Ia/Ib antigen expression. RJ-A3 is resistant to lysis by most Vgamma9/Vdelta2 lines and clones, even those lacking CD9-4/NKG2 and p58, p70 p140 KIR molecules. However, one Vgamma9/Vdelta2 line which can efficiently kill RJ-A3 do so in a TCR-dependent manner since killing is modulated by anti-TCR antibodies. The CDR3 sequences of the T cell clones amplified with Daudi and RJ-A3 reveal that some clones can be expanded with both lines while others are expanded preferentially with one or the other but not both. This indicates differences in the antigenic determinants of the two Burkitt's lines. The occurrence of this Raji variant line demonstrates that the stimulatory phenotype for Vgamma9/Vdelta2 cells can be acquired by some tumors independently of the loss of class I antigens and comforts the hypothesis of an anti-tumoral function for the Vgamma9/Vdelta2 T cell population.

Predominance of Vgamma9/Vdelta2 T lymphocytes in the cerebrospinal fluid of children with tuberculous meningitis: reversal after chemotherapy.

BACKGROUND: We analyzed the gammadelta T cell composition and responses in the peripheral blood and cerebrospinal fluid (CSF) of children affected by tuberculous meningitis (TBM) and in control children. MATERIALS AND METHODS: Peripheral blood and CSF samples were stimulated with different phosphoantigens and IL-2, and expansion of Vgamma9/Vdelta2 T cells assessed by FACS analysis. Vgamma9/Vdelta2 lines were obtained by culturing CSF or peripheral blood mononuclear cells (PBMC) in vitro with phosphoantigens and IL-2 for 2 months, and tested for proliferation and cytokine production in response to phosphoantigens. Vdelta2(D)Jdelta junctional sequence length was assessed by PCR. RESULTS: The repertoire of gammadelta T cells from the CSF of TBM patients was characterized by the predominance of Vgamma9/Vdelta2 T lymphocytes, which accounted for >80% of gammadelta T cells. Vgamma9/Vdelta2 cells from the CSF of TBM children responded to different synthetic and natural (mycobacterial) phosphoantigens and produced discrete amounts of IFN-gamma and TNF-alpha. The in vitro expansion of Vgamma9/Vdelta2 T cells from CSF and peripheral blood of TBM patients prominently decreased following chemotherapy, and similarly, the proportion of ex vivo unstimulated Vgamma9/Vdelta2 T cells in CSF of TBM patients decreased to levels detected in the CSF of control subjects. Vdelta2 CDR3 TCR analysis showed that the remaining Vdelta2 cells in the CSF of TBM patients were still polyclonal. CONCLUSIONS: These findings are consistent with an involvement of Vgamma9/Vdelta2 T cells in TBM. http://link. springer-ny.com/link/service/journals/00020/bibs/5n5p301. html

Primary lens luxation in the Chinese Shar Pei: clinical and hereditary characteristics.

A pedigree analysis of a family of 15 related Chinese Shar Peis was conducted. This pedigree analysis, including affected and nonaffected dams, sires and offspring, was compiled to document and characterize the occurrence, common clinical signs, and age of onset of primary lens luxation while suggesting a possible mode of inheritance in this breed. Of the five offspring from the mating of an affected dam to two unrelated affected males, 100% of offspring were affected with bilateral primary lens luxations. Of the four viable offspring from the mating of the same affected dam to an unrelated, unaffected male, two dogs (50%) were affected. The average age of onset of affected animals (seven) in this first generation was 4.9 years (range 3-6 years). The six dogs in the second generation of the same pedigree line were 2-years-old at examination with none of these animals affected at the time of this study. The most common presenting complaints were a unilateral change in ocular appearance (5 of 7 dogs) and subjective vision impairment (4 of 7 dogs). The most common clinical sign upon ophthalmic examination was iridodonesis (unilateral 4 of 7 dogs; bilateral 3 of 7 dogs) and the presence of an aphakic crescent (3 of 7 dogs). Gonioscopy and tonometry of severely affected eyes revealed a narrow or closed iridocorneal angle and ocular hypertension. This study suggests that primary lens luxation does occur in the Chinese Shar Pei, resembling the clinical condition (age of onset, clinical signs) previously described in the terrier breeds, the Border Collie, and the Tibetan Terrier. Application of the phenotypic findings in this study to a Mendelian genetic model of inheritance suggests that primary lens luxation in the Chinese Shar Pei is inherited as a simple autosomal recessive trait.

Control of self-reactive cytotoxic T lymphocytes expressing gamma delta T cell receptors by natural killer inhibitory receptors.

The majority of peripheral blood gamma delta T cells in human adults expresses T cell receptors (TCR) with identical V regions (V gamma 9 and V delta 2). These V gamma 9 V delta 2 T cells recognize the major histocompatibility complex (MHC) class I-deficient B cell line Daudi and broadly distributed nonpeptidic antigens present in bacteria and parasites. Here we show that unlike alpha beta or V gamma 9- gamma delta T cells, the majority of V gamma 9V delta 2 T cells harbor natural killer inhibitory receptors (KIR) (mainly CD94/NKG2A heterodimers), which are known to deliver inhibitory signals upon interaction with MHC class I molecules. Within V gamma 9V delta 2 T cells, KIR were mainly expressed by clones exhibiting a strong lytic activity against Daudi cells. In stark contrast, almost all V gamma 9V delta 2 T cell clones devoid of killing activity were KIR-, thus suggesting a coordinate acquisition of KIR and cytotoxic activity within V gamma 9V delta 2 T cells. In functional terms, KIR inhibited lysis of MHC class I-positive tumor B cell lines by V gamma 9V delta 2 cytotoxic T lymphocytes (CTL) and raised their threshold of activation by microbial antigens presented by MHC class I-positive cells. Furthermore, masking KIR or MHC class I molecules revealed a TCR-dependent recognition by V gamma 9V delta 2 CTL of ligands expressed by activated T lymphocytes, including the effector cells themselves. Taken together, these results suggest a general implication of V gamma 9V delta 2 T cells in immune response regulation and a central role of KIR in the control of self-reactive gamma delta CTL.

Patterns of phosphoantigen stimulation of human Vgamma9/Vdelta2 T cell clones include Th0 cytokines.

This paper examines functional properties of human Vgamma9/Vdelta2 T cell lines and clones generated by in vitro culture with synthetic and natural (mycobacterial) phosphoantigenic molecules. It confirms the broad reactivity of Vgamma9/Vdelta2 T cell lines and clones toward phosphoantigens. Optimal recognition of phosphoantigens by Vgamma9/Vdelta2 T cells required accessory cells to occur, but did not require specialized antigen presenting cells. However, species origin of the APC was irrelevant as proliferation of Vgamma9/Vdelta2 T cells occurred in the presence of syngeneic, allogeneic or xenogeneic APC and was not restricted to APC of particular tissue origin. Moreover antigen uptake and processing was not required for recognition by Vgamma9/ Vdelta2 cells, as evidenced by the ability of fixed APCs to present phosphoantigens. Similarly, the expression of classical MHC class I and class II molecules was not required for phosphoantigen recognition by gammadelta T cells. However, gammadelta T cell clones responded to stimulation by several cytokines including IL-12, IFNgamma and TNFalpha. Finally, Vgamma9/Vdelta2 T cell clones preferentially produced both IFN-gamma and IL-4 in response to PHA or TUBAg stimulation, revealing that a Th0 pattern of cytokine production is frequent among these cells.

CD28/CTLA4-B7 interaction is dispensable for T cell stimulation by mouse mammary tumor virus superantigen but not for B cell differentiation and virus dissemination.

B cells are the primary targets of infection for mouse mammary tumor virus (MMTV). However, for productive retroviral infection, T cell stimulation through the virally-encoded superantigen (SAG) is necessary. It activates B cells and leads to cell division and differentiation. To characterize the role of B cell differentiation for the MMTV life cycle, we studied the course of infection in transgenic mice deficient for CD28/CTLA4-B7 interactions (mCTLA4-H gamma 1 transgenic mice). B cell infection occurred in CTLA4-H gamma 1 transgenic mice as integrated proviral DNA could be detected in draining lymph node cells early after infection by polymerase chain reaction analysis. In mice expressing I-E, B cells were able to present the viral SAG efficiently to V beta 6+ T cells. These cells expanded specifically and were triggered to express the activation marker CD69. Further stages of progression of infection appeared to be defective. Kinetics experiments indicated that T and B cell stimulation stopped more rapidly than in control mice. B cells acquired an activated CD69+ phenotype, were induced to produce IgM but only partially switched to IgG secretion. Finally, the dissemination of infected cells to other lymph nodes and spleen was reduced and the peripheral deletion of V beta 6+ T cells was minimal. In contrast, in mice lacking I-E, T cell stimulation was also impaired and B cell activation undetectable. These data implicate B7-dependent cellular interactions for superantigenic T cell stimulation by low-affinity TCR ligands and suggest a role of B cell differentiation in viral dissemination and peripheral T cell deletion.