Comparison of T cell receptor alpha, beta, and gamma gene rearrangement and expression in T cell acute lymphoblastic leukemia.

We have analyzed the configuration of the T cell receptor (TCR) alpha gene using newly developed genomic joining region (J alpha) probes, which cover approximately 80 kb of the J alpha region upstream from the constant region in 19 patients with T cell acute lymphoblastic leukemia (T-ALL) and in three CD3- leukemic T cell lines (HSB2, CEM, and MOLT4). In parallel, transcription of the TCR-alpha, beta, and gamma genes was examined in 11 of these patients and in the T cell lines. All T-ALL and the three T cell lines exhibited both TCR-gamma and beta gene rearrangements. 8 of 10 T-ALL and all T cell lines expressed TCR-gamma transcripts. All samples tested expressed both TCR-beta and CD3-gamma transcripts. TCR alpha transcripts were only observed in CD3+ T-ALL but not in CD3- T-ALL or the CD3- cell lines. Among the CD3+ T-ALL, eight had TCR-alpha gene rearrangements. In addition, TCR-alpha gene rearrangements were detected in one CD3- T-ALL and all three T cell lines. These leukemic cells may represent a transient stage between rearrangement and expression and provide an opportunity for analyzing the mechanism regulating the expression of the TCR-alpha gene.

T cell receptor delta gene rearrangements in acute lymphoblastic leukemia.

Using a newly isolated cDNA clone encoding the TCR-delta gene and genomic probes, we have analyzed T cell receptor (TCR) delta gene rearrangement in 19 patients with T cell acute lymphoblastic leukemia (T-ALL) and 29 patients with B-precursor ALL. Five out of seven CD3- T-ALL and 4 of 12 CD3+ T-ALL showed bi-allelic rearrangements of the TCR-delta gene. In three CD3+ patients, a single allelic TCR-delta gene rearrangement was observed with rearrangement of the TCR-alpha gene on the other allele. In five CD3+ patients with bi-allelic rearrangements of the TCR-alpha gene, the TCR-delta gene locus was deleted. Transcription of the TCR-delta gene was also analyzed in six T-ALL. Five patients expressed TCR-delta transcripts. Only one T-ALL, presumably derived from the most immature T lineage cells, did not have TCR-delta transcripts, but expressed TCR-gamma and 1.0-kb truncated TCR-beta transcripts. In B-precursor ALL, 20 patients (69%) showed rearrangements of the TCR-delta gene. The frequency of TCR-delta gene rearrangement was higher than TCR-alpha (59%), gamma (52%), or beta (31%) genes. These findings suggest that TCR-alpha gene rearrangements may take place after rearrangements of the TCR-delta gene with concomitant deletion of rearranged TCR-delta genes in T cell differentiation. Among leukemic cells of B lineage, the TCR-delta gene is the earliest rearranging TCR gene, followed by TCR-gamma and beta gene rearrangements.

The isolation of the human delta chain gene and its expression in normal T cells and T cell leukemias.

In this paper we describe a gene that lies 85 kb 5' of the constant region of the human alpha chain and some 30-50 kb 3' of the alpha chain V region (H. Griesser, unpublished observation). This gene undergoes somatic recombination and is transcribed in thymocytes, PHA-stimulated peripheral blood T cells, and some T cell leukemic cell lines. Sequence analysis revealed that the gene has a structure similar to that of immunoglobulin and other T cell antigen receptor genes. Comparison of the sequence to a mouse gene found in a similar location revealed 80% homology at the protein level. Recently, M. Davis and A. Weiss (personal communication) have demonstrated that the protein product of the mouse gene is the delta chain gene. Thus, the gene described in this paper represents the human homolog of the delta chain gene of the T cell antigen receptor.

An alternative method for T-cell receptor repertoire analysis: clustering of human V-beta subfamilies selected in responses to staphylococcal enterotoxins B and E.

We have designed a convenient procedure for the analysis of V beta repertoire expression in polyclonal T-cell populations. In this procedure T-cell RNA is converted to cDNA, polydC-tailed with terminal deoxynucleotidyl transferase and submitted to one-side specificity PCR amplification with a constant region oligonucleotide primer. The amplified material is then analysed by reverse spot-test hybridization: after 32P-labelling, the amplification product is put to hybridize on a membrane where specially designed V beta subfamily-specific probes are immobilized. The radioactivity fixed on each probe can then be easily quantified and the signal obtained is directly proportional to the initial amount of homologous RNA. We applied this technique to the study of V beta gene selection following T-cell stimulation by staphylococcal enterotoxins B and E. We show that with these toxins two almost non-overlapping sets of T-cells are recruited and that this selection is likely to be dependent on specific amino acid residues shaping the fourth complementarity determining region of the TCR-beta chain. These residues constitute two tandemly-conserved tripeptide sequences (Asp39Pro40Gly41)-(Val69Ser70Arg71) and (Arg66Phe67Ser68)-(Asp88Ser89Ala90) in the SEB- and the SEE-responsive V beta gene clusters respectively.

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.

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.

Abnormal T cell receptor V beta gene expression in the peripheral blood and synovial fluid of rheumatoid arthritis patients.

OBJECTIVE: The aim of this study was to assess T cell receptor V beta-gene expression in the peripheral blood and synovial fluid of rheumatoid arthritis patients. METHODS: Cytometric analysis was performed on peripheral blood and synovial fluid lymphocytes from 12 patients using a restricted set of V beta-specific monoclonal antibodies (to V beta 5.1-3, V beta 6.7 and V beta 8). In 5 patients the expression of the V beta 1 through V beta 20 gene families was also analysed, using a recently described method based on a one-side-specificity polymerase chain reaction coupled to reverse dot hybridization. RESULTS: Cytometric analysis failed to show any consistent difference in the expression of V beta 5, 6 and 8 between the two compartments on the one hand, or between the peripheral blood of normal individuals and patients on the other hand. The PCR/dot hybridization method did not demonstrate a significant difference in the V beta repertoires between peripheral blood and synovial fluid samples from arthritis patients. However, in all patients the V beta 6, 13 and/or 14 families were expressed to a high level, so that these families frequently represented over 40% of the V beta 1-20 repertoire in both compartments, instead of the approximately 20% seen in normal peripheral blood samples. CONCLUSION: We conclude that V beta 6, 13 and 14 are overexpressed in both the peripheral blood and synovial fluid of rheumatoid arthritis patients compared to normal samples.

Effects of Ca(II) ions on Cu(II) ion-phytic acid interactions.

In vitro interactions among phytic acid (PA), Cu(II) ions, and Ca(II) ions were examined as functions of PA:Cu(II):Ca(II) molar ratios and pH. Ca(II) ions competed with Cu(II) ions for binding by the soluble phytate species for PA:Cu(II) molar ratios ranging from 10:1 to 1:6 and pH values in the 2.4-5.9 range. At pH values where precipitation occurred, Ca(II) ions potentiated Cu(II) ion binding by the precipitated phytate species for PA:Cu(II) molar ratios of 10:1 to 1:3. At lower PA:Cu(II) molar ratios, Ca(II) ions competed with Cu(II) ions for binding by the precipitated phytate species. Compositions of the precipitated copper-calcium phytates are reported.

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.

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.

Low gastric hydrochloric acid secretion and mineral bioavailability.

Young infants and approximately 30% of the elderly have low secretion of hydrochloric acid by gastric parietal cells. It has been established that low hydrochloric acid secretion can lead to decreased absorption of ferric iron. Conflicting results have been obtained in clinical studies of the effects of intraluminal gastric pH values on calcium absorption. The results of an in vitro study suggest that the chemical form of the ingested calcium and the presence of protein may influence whether high intraluminal gastric pH values affect resultant calcium solubilities in the small intestine. The effects of low hydrochloric acid secretion on zinc absorption have not been ascertained. The results of an in vitro study indicate that high intraluminal gastric pH values would not affect resultant zinc solubilities in the small intestine following pancreatin digestion of soy protein isolate supplemented with calcium and/or zinc. Considering that the diets of many elderly contain primarily plant foods and that soy protein isolate formulas are commonly fed to infants, further research is especially needed to determine the effects of low hydrochloric acid secretion on mineral bioavailabilities from high fiber and phytate containing plant foods.

Ophthalmic cyclosporine in equine keratitis and keratouveitis: 11 cases.

Topical cyclosporine A was safely used in a series of 11 cases of equine keratitis and keratouveitis and appeared to be an effective anti-inflammatory agent in 9 cases. The clinical diagnoses included interstitial keratouveitis, endotheliitis, multifocal punctate keratopathy and a melting stromal ulcer. In most cases, the presence or absence of insidious bacterial infection was not conclusively determined. Topical cyclosporine A had no deleterious effects in this series of cases. The authors suggest that topical cyclosporine in both aqueous and lipid base vehicles should be investigated and evaluated as an alternative mode of achieving ocular immunosuppression.

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.

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.

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.

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.

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.