Chemokine expression and leucocyte infiltration in Sjogren's syndrome

OBJECTIVE: To investigate the expression and source of chemokines in minor salivary gland biopsies (MSGs) in patients with Sjogren's syndrome (SS). METHODS: Immunohistochemical analysis was used to determine the pattern of chemokine expression in MSGs from patients with (n=6) and without (n=5) SS, as well as to examine the phenotype of both resident and infiltrating cells expressing chemokines. RESULTS: Significant differences in the number of infiltrating mononuclear (MN) cells in patients with and without SS were noted. Ductal epithelial cells of SS biopsies expressed significantly increased levels of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, interleukin-8 (IL-8) and RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted). Biopsies from patients with SS showed that MIP-1beta was expressed by 51% of infiltrating cells, while 41% expressed MIP-1alpha, whereas 22 and 7% expressed RANTES and IL-8, respectively. CONCLUSION: Chemokines expressed by ductal epithelial cells may attract circulating leucocytes, in particular CD4+ T cells, towards the site of inflammation, thereby orchestrating the influx of MN cells characteristically seen in MSGs in SS. Chemokines may be induced directly by a putative triggering agent for SS, or secondary to the release of pro-inflammatory cytokines produced by epithelial cells. These findings further implicate epithelial cells as playing a major role in the pathogenesis of SS and implicate chemokines in the leucocyte recruitment in this setting.      

Coagulation factor activity and clinical bleeding severity in rare bleeding disorders: results from the European Network of Rare Bleeding Disorders

Summary. Background: The European Network of Rare Bleeding Disorders (EN‐RBD) was established to bridge the gap between knowledge and practise in the care of patients with RBDs. Objectives: To explore the relationship between coagulation factor activity level and bleeding severity in patients with RBDs. Patients/Methods: Cross‐sectional study using data from 489 patients registered in the EN‐RBD. Coagulation factor activity levels were retrieved. Clinical bleeding episodes were classified into four categories according to severity. Results: The mean age of patients at data collection was 31 years (range, 7 months to 95 years), with an equal sex distribution. On linear regression analysis, there was a strong association between coagulation factor activity level and clinical bleeding severity for fibrinogen, factor (F) X, FXIII, and combined FV and FVIII deficiencies. A weaker association was present for FV and FVII deficiencies. There was no association between coagulation factor activity level and clinical bleeding severity for FXI. The coagulation factor activity levels that were necessary for patients to remain asymptomatic were: fibrinogen, > 100 mg dL^?1; FV, 12 U dL^?1; combined FV + VIII, 43 U dL^?1; FVII, 25 U dL^?1; FX, 56 U dL^?1; FXI, 26 U dL^?1; FXIII, 31 U dL^?1. Moreover, coagulation factor activity levels that corresponded with Grade III bleeding were: undetectable levels for fibrinogen, FV and FXIII, < 15 U dL^?1 for combined FV + VIII; < 8 U dL^?1 for FVI; < 10 U dL^?1 for FX; and < 25 U dL^?1 for FXI. Conclusions: There is a heterogeneous association between coagulation factor activity level and clinical bleeding severity in different RBDs. A strong association is only observed in fibrinogen, FX and FXIII deficiencies.     

In vitro interactions of PGE and cAMP with murine and human erythroid precursors

Addition of prostaglandins of the E series (PGE1, PGE2) in methylcellulose cultures of murine marrow results in a dose-dependent inhibition of the cloning efficiency of both BFU-E and CFU-C. However, CFU-E growth is unaffected. The inhibitory action of PGE is progressively overcome by increasing amounts of colony-stimulating factor (CSF), and with some limitations, also of erythropoietin (Ep). Addition of PGF2 alpha' associated or not with indomethacin, does not exert any significant effect on these hemopoietic precursors. In an attempt to unvail the mechanism(s) underlying these phenomena, dibutyryl-cyclic AMP (db-cAMP), theophylline (an inhibitor of phosphodiesterase), or theophylline + PGE were plated at various concentrations. Both db-cAMP and theophylline induce an inhibitory influence on both BFU-E and CFU-C growth, which mimicks that by PGEs; additionally, theophylline potentiates the inhibitory action of PGE1. In all these studies, the CFU-E number was not significantly modified. PGE action on BFU-E proliferation is clearly species-dependent, since PGE1 addition to human marrow methylcellulose cultures induces a significant enhancement of the number of both BFU-E and CFU-E derived colonies. This action was abolished upon removal of adherent cells, thus suggesting that PGE1 evokes a release of factor(s) enhancing human erythroid colony growth by adherent cells.     

Expression of phosphorylcholine-specific B cells during murine development

The TEPC 15 (T15) clonotype, a putatively germline antibody specificity, does not appear in the neonatal B-cell repertoire until approximately 1 wk of age. This report extends this observation by the demonstration that (a) the T15 clonotype follows similar kinetics of appearance in germfree as well as conventionally-reared mice; (b) maternal influences and genetic background play a minor role in the development of the T15 clonotype since CBFI neonates raised by C57BL/6 or BALB/c mothers acquire the T15 clonotype at the same time in ontogeny as BALB/c neonates; (c) the lack of phosphorylcholine (PC)-specific B cells shortly after birth is reflected in a dearth of PC-binding cells in the neonate as well; and (d) no PC-specifc B cells are found in 19-day fetal liver or in bone marrow until 7 days of life, coincident with their appearance in the spleen. These findings, along with a previous report that PC-specific splenic B cells are tolerizable as late as day 10 after birth, confirm the invariant, late occurrence of the T15 clonotype and support a highly- ordered, rigorously predetermined mechanism for the acquisition of the B- cell repertoire. The results are discussed in light of other studies on the ontogeny of B-cell specificity, and in terms of the implications on the mechanism by which antibody diversity is generated.     

The coagulant-active phospholipid content is a major determinant of in vivo thrombogenicity of prothrombin complex (Factor IX) concentrates in rabbits

In vitro evaluation of prothrombin complex concentrates in a thrombin generation assay, using DAPA and purified components of the prothrombinase complex, demonstrated significant levels of coagulant- active "phospholipid replacing" activity. Quantification of this activity showed a significant correlation (r = 0.8747, p less than 0.01) with thrombogenicity measured in vivo in a stasis model in rabbits. Extracted lipid material retained full phospholipid replacing activity in the vitro assay. Thin-layer chromatographic characterization confirmed the presence of phospholipids with known coagulant activity in vitro. In vivo, the extracted material was nonthrombogenic but augmented the thrombogenicity of purified factor Xa. Substitution of a synthetic coagulant-active phospholipid (phosphatidylcholine-phosphatidylserine lipid vesicles) for the extracted phospholipid produced a similar augmentation of a factor-Xa- induced thrombogenicity in vivo. It is concluded that the coagulant- active phospholipid content of prothrombin complex concentrates is a major determinant of thrombogenicity but requires the presence of activated clotting factors for its expression in vivo.     

Prevalence and clinical significance of antineutrophil cytoplasmic antibodies in Churg-Strauss syndrome

OBJECTIVE: Churg-Strauss syndrome (CSS) is classified among the so-called antineutrophil cytoplasmic antibody-associated systemic vasculitides (AASVs) because of its clinicopathologic features that overlap with the other AASVs. However, while antineutrophil cytoplasmic antibodies (ANCAs) are consistently found in 75-95% of patients with Wegener's granulomatosis or microscopic polyangiitis, their prevalence in CSS varies widely and their clinical significance remains uncertain. We undertook this study to examine the prevalence and antigen specificity of ANCAs in a large cohort of patients with CSS. Moreover, we evaluated the relationship between ANCA positivity and clinicopathologic features. METHODS: Immunofluorescence and enzyme-linked immunosorbent assay were used to determine the presence or absence of ANCAs in 93 consecutive patients at the time of diagnosis. The main clinical and pathologic data, obtained by retrospective analysis, were correlated with ANCA status. RESULTS: ANCAs were present by immunofluorescence in 35 of 93 patients (37.6%). A perinuclear ANCA (pANCA) pattern was found in 26 of 35 patients (74.3%), with specificity for myeloperoxidase (MPO) in 24 patients, while a cytoplasmic ANCA pattern, with specificity for proteinase 3, was found in 3 of 35 patients (8.6%). Atypical patterns were found in 6 of 30 patients with anti-MPO antibodies (20.0%). ANCA positivity was associated with higher prevalences of renal disease (51.4% versus 12.1%; P < 0.001) and pulmonary hemorrhage (20.0% versus 0.0%; P = 0.001) and, to a lesser extent, with other organ system manifestations (purpura and mononeuritis multiplex), but with lower frequencies of lung disease (34.3% versus 60.3%; P = 0.019) and heart disease (5.7% versus 22.4%; P = 0.042). CONCLUSION: ANCAs are present in approximately 40% of patients with CSS. A pANCA pattern with specificity for MPO is found in most ANCA-positive patients. ANCA positivity is mainly associated with glomerular and alveolar capillaritis.     

Natural interferon-alpha versus its combination with 6-methyl- prednisolone in the therapy of type II mixed cryoglobulinemia: a long- term, randomized, controlled study

Type II mixed cryoglobulinemia (MC) is an often progressive vasculitis characterized by circulating cold-precipitable proteins that usually consists of polyclonal IgG and monoclonal IgM kappa with rheumatoid factor (RF) activity. Its etiology is unknown, although recent evidence strongly suggests that hepatitis C virus (HCV) plays a major role. Plasmapheresis, corticosteroids, and cytotoxic drugs have been used in the therapy of MC patients. Recently, favorable results with recombinant interferon-alpha (rIFN alpha) have been reported. To further assess its effectiveness, we studied the effects of natural human interferon-alpha (nIFN alpha), alone and in combination with 6- methyl-prednisolone (PDN), in a prospective, randomized, controlled trial in patients with symptomatic MC. Sixty-five patients were enrolled onto the trial, 52 (80%) of whom presented serum anti-HCV antibodies and specific genomic RNA sequences. Fifteen patients received nIFN alpha (3 MU) intramuscularly (IM) three times weekly, whereas 17 patients also received 16 mg/d of PDN orally on non-IFN days. Moreover, 18 patients received 16 mg/d of PDN only, and 15 were untreated. Treatment was discontinued after 1 year and patients were monitored for 8 to 17 months (mean, 13). A complete response was achieved in eight of 15 patients (53.3%) treated with nIFN alpha and nine of 17 (52.9%) treated with nIFN alpha plus PDN, as compared with three of 18 patients (16.7%) who received PDN only (P < .05) and one of 15 (6.7%) untreated controls (P < .01). Partial response occurred in two of 15 (13.3%) patients treated with nIFN alpha, three of 17 (17.6%) who received nIFN alpha plus PDN, one of 18 (5.5%) who received PDN only, and one of 15 (6.7%) controls. A complete response in six patients (66.7%) was achieved within 3 months in the group that received nIFN alpha plus PDN, as compared with two patients (25%) of those who received nIFN alpha alone (P < .02). In anti-HCV-positive patients, the clinical response occurred in step with reduced or undetectable levels of HCV RNA and transaminase normalization. Quantification of circulating HCV RNA represented a good predictive response marker. The probability of relapse within 3 months after treatment was 100% (three of three patients) and 75% (six of eight patients), respectively, in patients who received PDN alone or nIFN alpha alone as compared with none of those who received nIFN alpha plus PDN (P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Microaggregate counts in frozen-preserved erythrocytes: effects of washing in three blood processors and filtration

Since microaggregates have been implicated in posttransfusion pulmonary insufficiency, their elimination has become an active concern in blood transfusion. Various types of filters, as well as frozen-preserved erythrocytes, have been used to provide blood relatively low in microaggregates. We have counted particles in frozen-stored blood before deglycerolization, after washing in each of three cell processing systems, and after filtration through a 40-micrometer filter. Washing frozen erythrocytes reduced the total particle counts by an average of 89%. Slight differences were found among the three blood processors with respect to particle removal. Passing washed blood through a 40-micrometer filter did not result in significant further reduction in particle counts. Hence, the use of such filters in a frozen-preserved blood system is not warranted.