CD3+ leukemic large granular lymphocytes utilize diverse T-cell receptor V beta genes

CD3+ large granular lymphocyte (LGL) leukemia is a disease of unknown etiology characterized by clonal proliferation of T cells that usually express T-cell receptor (TCR) alpha beta heterodimers. The purpose of this study was to identify the variable (V), joining (J), and diversity (D) region TCR beta-chain genes expressed by CD3+ LGL leukemic cells in an attempt to gain insights into the etiology of this disorder. Twelve patients with LGL leukemia were studied, including seven with both LGL leukemia and rheumatoid arthritis (RA). RA is also a disease of unknown etiology that occurs frequently in patients with LGL leukemia. Clonally expanded T cells that express specific TCR V beta genes have been identified in fluid and tissue specimens from the joints of patients with RA. In this study, V beta expression was determined by PCR using a panel of 22 unique V beta primers to amplify cDNA prepared from peripheral blood mononuclear cells (PBMC). A dominant V beta gene product was readily apparent in all patients. To confirm that the dominant V beta gene originated from a clonal expansion, DNA fragments corresponding to the dominant V beta genes were subcloned into plasmids and independently isolated recombinants were sequenced. V-D-J region sequences that occurred repeatedly indicated clonality. The V beta and J beta genes expressed by the leukemic cells showed a pattern of distribution that followed the frequency with which these genes are represented in the peripheral blood. The residues corresponding to the third complementarity-determining region of the TCR beta chain were different in all cases. A specific pattern of VDJ usage was not identified for those patients with both LGL leukemia and RA; however, utilization of V beta-6 by LGL clones (N = 3) was observed only in the setting of RA. These data suggest that leukemic CD3+ LGL cells have been clonally transformed in a random fashion with respect to the TCR beta chain.     

Heparin oligosaccharides bind L- and P-selectin and inhibit acute inflammation

Initial attachment of leukocytes to the vessel wall at sites of inflammation is supported by a family of carbohydrate-binding adhesion molecules called the selectins. Selectin ligands include sialyl-Lewis x (sLex, Neu5Ac alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc--) and related structures. We report here that defined heparin oligosaccharides interact with the selectins. Heparin chains containing four or more monosaccharide residues inhibited the function of L- and P-selectin, but not E-selectin, in vitro. In a competition enzyme-linked immunosorbent assay measuring inhibition of solution-phase selectin-Ig fusion proteins (selectin-Ig) binding to immobilized bovine serum albumin-sLex neoglycoprotein, a heparin-derived tetrasaccharide mixture inhibited 50% of L- and P-selectin-Ig binding (IC50) at 200 +/- 40 mumol/L and 850 +/- 110 mumol/L, respectively. A single hexasulfated tetrasaccharide (delta UA2S alpha 1-4GlcNS6S alpha 1-4IdoA2S alpha 1- 4GlcNS6S) was particularly active against L- and P-selectin-Ig (IC50 = 46 +/- 5 mumol/L and 341 +/- 24 mumol/L). By comparison, the tetrasaccharide sLex was not inhibitory at concentrations up to 1 mmol/L. In cell adhesion assays, heparin tetrasaccharides reduced binding of neutrophils to COS cells expressing P-selectin but not to COS cells expressing E-selectin. They also blocked colon cancer cell adhesion to L- and P-selectin but not E-selectin. In a model of acute inflammation, intravenously administered heparin tetrasaccharides diminished influx of neutrophils into the peritoneal cavities of thioglycollate-treated mice. We conclude that heparin oligosaccharides, including non-anticoagulant tetrasaccharides, are effective L- and P- selectin inhibitors in vitro and have anti-inflammatory activity in vivo.     

The activation of the contact phase of coagulation by physiologic surfaces in plasma: the effect of large negatively charged liposomal vesicles

The endogenous, negatively charged surface that induces activation of the contact coagulation factors was investigated in plasmas taken from women in late pregnancy and control subjects of child-bearing age. The plasmas from the two groups of subjects were incubated at 4 degrees C for 24 hours either in plastic or in glass tubes and the factor VII coagulant activity (VIIc) was assayed in the treated plasmas. The activation of factor VII under these conditions involves the generation of enzymes derived from factor XII (XIIa). The contact surface is rate- limiting for the activation of factor VII in the plasmas in both groups of subjects and can be supplemented by large multilamellar liposomal vesicles carrying the appropriate density of negative charge. The size of these vesicles is within the range of sizes of the large lipoprotein particles (chylomicrons, very low and intermediate-density lipoproteins). The relationship between the density of negative charge on the liposomal vesicles and VIIc was similar in the late pregnancy and the control plasmas incubated in plastic tubes. At a saturating density of negative charge the observed relative VIIc was similar in both sets of plasmas. The incubation of late pregnancy or control plasma in plastic tubes in the presence of sodium stearate caused VIIc to increase with increasing concentration of the added fatty acid. These results suggest that large lipoprotein particles carrying the appropriate free fatty acid at a sufficient density of negative charge could provide the contact surface that induces the generation of factor XIIa and the subsequent activation of factor VII. Moreover, plasmas from women in late pregnancy have a higher concentration of potential surface and a higher density of negative charge than the plasmas from nonpregnant women.     

Production of cytokines by bone marrow cells obtained from patients with multiple myeloma

Previous work with continuously cultured multiple myeloma lines suggested that cytokine production by tumor cells may mediate some of the medical complications of this disease. To further investigate this issue, we assayed freshly obtained bone marrow (BM) cells from myeloma patients for the in vitro production of cytokines and the presence of cytokine RNA. Production of cytokine protein was assessed by bioassays with the aid of specific neutralizing anticytokine antibodies. These assays detected interleukin-1 (IL-1) and tumor necrosis factor (TNF) secretion by myeloma BM cells, which was significantly greater than secretion from similarly processed BM cells of control individuals. In contrast, lymphotoxin and interleukin-2 (IL-2) production could not be detected. The levels of IL-1 and TNF produced in vitro peaked at 24 hours of culture and correlated with stage and the presence (or absence) of extensive osteolytic bone disease. Northern blot analysis demonstrated the presence of IL-1 beta and TNF RNA in uncultured myeloma BM cells but no detectable IL-1 alpha or lymphotoxin RNA. In addition, the amount of cytokine RNA correlated with protein production, being significantly greater in patients' BM cells than in control marrow. These data suggest a role for IL-1 beta and/or TNF in the pathophysiology of multiple myeloma and argue against a role for lymphotoxin or IL-2.     

Platelet gel in cutaneous radiation dermatitis

Introduction

Radiotherapy, alone or in combination with chemotherapy and/or surgery, is a fundamental and irreplaceable method of treating tumours. Nonetheless, although the technological advances made during recent years and the associated improvements in this type of treatment have reduced the incidence of complications, 5?C15?% of patients still experience damage to the healthy tissues exposed to radiation. Cutaneous and mucosal lesions are severe collateral effects of radiotherapy that have an enormous impact on a patient??s quality of life. Unfortunately, however, the efficacy of conventional treatments, while demonstrably useful in acute lesions, remains disputed in chronic cases. Nevertheless, numerous studies and clinical findings have demonstrated that topical, non-transfusional plasma-rich platelet gel is able to accelerate the regeneration and repair of tissues through the action of the various growth factors contained within the alpha granules of platelets. We therefore set out to evaluate the efficacy of autologous platelet gel, chosen for its limited cost and ease of preparation, in chronic cutaneous radiation dermatitis.

Methods

??Home-made?? platelet gel was produced by treating platelets with autologous thrombin. The safety of the product was ensured by microbiological tests. The autologous platelet gel was applied topically once a week, for a mean duration of 35?days, to chronic third- and fourth-degree (European Pressure Ulcer Advisory Panel classification and Common Terminology Criteria for Adverse Events score) cutaneous radiation dermatitis in a group of ten patients previously treated for moderate-to-high grade (histology G2-G3) limb sarcoma by tumour excision and post-surgical radiotherapy (dose 50?C64?Gy). The radiation dermatitis had appeared at different intervals after treatment and had all proved resistant to conventional treatments.

Results

The autologous platelet gel was found to be successful in seven out of the ten patients treated. The various phases of the healing process were observed in all cases. Platelet gel application was suspended in three patients: in one patient after one application due to tumour progression, in another patient after two applications due to development of distant metastases and in the third after six applications with only partial tissue response. At 5-year follow-up, six of the seven successfully treated patients remained free of both disease and lesion, while the remaining patient, the eldest, had passed away in the interim due to extraneous causes.

Conclusion

Platelet gel treatment could therefore be used to bring about healing in chronic cutaneous radiation dermatitis, lending itself to better patient compliance and a favourable cost/benefit ratio, due to a reduction in the number of medications and hospital visits required.     

Association of vitiligo with HLA-A2: a meta-analysis

BACKGROUND: Linkage and association studies suggest that the human leucocyte antigen (HLA) region may be involved in the genetic susceptibility of vitiligo. HLA-A2 has been reported to be associated with vitiligo in some, but not all, studies. OBJECTIVE: To identify sources of the heterogeneity among studies and to quantify effect estimates, we examined the association of HLA-A2 with vitiligo in a meta-analysis of all observational studies comparing the frequencies of HLA-A2 between vitiligo individuals and controls during 1966-2005. METHODS: The summary odds ratio (OR) was calculated by using a fixed- or a random-effects model. Meta-regression analysis was undertaken to investigate the effects of study characteristics on the pooled OR. RESULTS: Eleven case-controlled studies fulfilled our inclusion criteria. The studies identified a total of 777 patients and 4820 controls. Meta-analysis showed a significantly increased frequency of HLA-A2 in vitiligo among cases [OR = 2.07, 95% confidence interval (CI) 1.67-2.58]. Heterogeneity was explained by the quality of the study and the ethnic background of the participants. Meta-regression analysis further showed that the percentage of familial vitiligo among the subjects had a significant effect on the pooled OR (P = 0.008). No study had a significant effect on the pooled OR and no publication bias presented in the studies analysed (P = 0.688). CONCLUSION: These findings strongly suggest an association between HLA-A2 and vitiligo.