Long-Term Follow-Up of the Changes in Circulating Cytokines, Soluble Cytokine Receptors, and White Blood Cell Subset Counts in Patients with Rheumatoid Arthritis (RA) After Monoclonal Anti-TNFα Antibody Therapy

To investigate the mechanism of the long-lasting efficacy of chimeric monoclonal anti-TNF antibody (cA2) therapy for rheumatoid arthritis (RA), eight patients with refractory RA were treated with a single infusion of cA2 and the changes in circulating cytokines (IL-1, IL-6, TNF, and IL-10), soluble cytokine receptors (TNF-RI, RII, and sIL-6R) and peripheral white blood cell (WBC) subset counts were followed up long-term (12 weeks) after cA2 therapy in them. Significant clinical responses (>20% improvement according to Paulus' criteria) were observed just after cA2 infusion and lasted more than 4 weeks in all patients, as reported elsewhere. Moreover, five of the eight patients showed prolonged clinical responses (>12 weeks). The elevated serum IL-6 and sTNF-RI (or RII) levels before treatment rapidly decreased after treatment. The serum IL-10 levels also significantly elevated before treatment. The elevations of serum IL-10 levels were augmented after treatment and stayed higher than the baseline in four patients with prolonged clinical responses. No significant TNF, IL-1 and -, or sIL-6R were detected in the sera of the patients before treatment and during the whole study period. On the other hand, peripheral lymphocytes as well as total WBC and neutrophils increased for 4 weeks after treatment. However, thereafter, only the lymphocyte count decreased gradually and stayed below the baseline long-term (12 weeks). FACS analysis revealed the predominance of T lymphocytes in the decrease in lymphocyte counts. These results suggest that the augmentation of IL-10 production and the decrease in T cells might partly contribute to the long-lasting efficacy of cA2 treatment in RA.     

Granulocytic sarcoma presenting with lymph node infarction at disease onset

A completely infarcted lymph node is an unusual event. However, lymph node infarction should alert the pathologist to the considerable likelihood of malignant lymphoma. We report two unusual cases of acute myeloid leukemia presenting with granulocytic sarcoma at disease onset with a lymph node lesion exhibiting extensive lymph node infarction. The infarcted tissue contained numerous eosinophilic cell ghosts. There were some islands of degenerated, pyknotic medium-sized nuclei resembling lymphoblasts present in the necrotic area. By immunohistochemistry, these medium sized cells were CD3-, CD20-, CD34+, CD43+, CD45RO-, CD68-, CD79a- and myeloperoxidase+ in both cases. Differentiation of granulocytic sarcoma from malignant lymphomas is important for adequate therapy. The present cases indicate that granulocytic sarcoma should be added to the list of differential diagnoses for lymph node infarction.     

Tumorous necrotic nodule in the liver: unexpected effect of the microwave tissue coagulator.

The microwave tissue coagulator (MTC) is used in hepatectomy because it provides excellent haemostasis during the procedure. A 59 year old man underwent partial hepatic lobectomy with MTC, for metastasis from colon cancer. A tumorous necrotic nodule was discovered in the liver. The nodule measured 2.5 cm at its largest diameter. Microscopically, it showed extensive coagulation necrosis and massive sinusoidal dilatation. To date, such a necrotic mass clinically mimicking neoplasm has not been reported as a complication of hepatectomy using MTC. Although it is unknown how the rounded necrotic nodule was formed in this case, clinicians should be aware of this phenomenon to avoid unnecessary operations. Likewise, pathologists should recognise such histological changes and review the clinical history of the patient when coagulation necrosis with massive sinusoidal dilatation is observed in a biopsy or hepatectomy specimen.     

Development of a PCR method for rapid identification of new Streptococcus mutans serotype k strains

In a previous study, we isolated and characterized a new serotype k of Streptococcus mutans from human blood and oral cavities. Analysis of the genes involved in biosynthesis of the serotype-specific polysaccharide of serotype k strains revealed that the serotype k-specific nucleotide alignment was commonly present in the 5' region of the rgpF gene (350 bp from the initial sequence) compared to the reference strains, and then a method for rapid identification of serotype k strains was developed by use of PCR with primers designed on the basis of the sequence of the variable region. PCR assays with primers specific for amplification of serotype k strains showed a negative reaction with serotype c, e, and f strains and a positive reaction with serotype k strains, with the sensitivity for identification of the serotype k strains shown to range from 5 to 50 cells. Next, the frequency of positive reactions for serotype k-specific primers was surveyed with DNA taken from saliva samples from 200 subjects (2 to 18 years of age), and 10 of those showed a positive reaction, which was higher than the frequency in our previous survey with a serological method. In addition, all saliva samples from subjects with serotype k strains in our previous study were shown to be positive with the serotype k-specific primers. These results indicate that this new PCR method is effective for identification of subjects with S. mutans serotype k.     

Evaluation of Intestinal Protozoan Morphology in Human Fecal Specimens Preserved in EcoFix: Comparison of Wheatley’s Trichrome Stain and EcoStain

As a result of disposal problems related to the use of mercury compounds, many laboratories have switched from mercuric chloride-based Schaudinn’s and polyvinyl alcohol (PVA) stool preservatives to other, non-mercury-based preservatives. A comparison of organism recoveries and morphologies of the intestinal protozoa was undertaken with PVA containing the EcoFix zinc-based Schaudinn’s preservative (Meridian Diagnostics, Inc.); both Wheatley’s modification of Gomori’s trichrome stain (WT) and EcoStain (ES) were used to stain 51 human fecal specimens. Morphology, clarity of nuclear and cytoplasmic detail, overall color differences, and the ease or difficulty in detecting intestinal protozoa in fecal debris were assessed for the two permanent stained smears. Overall, organism morphology of the intestinal protozoa stained with WT and that of protozoa stained with ES were not equal in nuclear and cytoplasmic detail or range of color. However, the same organisms were identified in stained fecal smears with either WT or ES, with the exception of situations in which organism numbers were characterized as rare. Included were 67 protozoan challenges (number of organisms): Entamoeba histolytica-Entamoeba dispar (5), Entamoeba coli (9), Entamoeba hartmanni (6), Endolimax nana (12), Iodamoeba bütschlii (8), Blastocystis hominis (19), Giardia lamblia (6), Dientamoeba fragilis (2), yeast (2), and leukocytes (2). Five specimens were negative for parasites but contained fecal debris that was compared for morphologic detail and color range. The ES produces a more gray-green monotone with very little pink or red tone; contrast among the various colors is less than that seen with WT. Stain intensity for all organisms was acceptable, and there were no problems with stain deposition. The quality of the protozoan morphology with ES was often comparable to that with WT (36 of 67 [53.7%]) and, in some cases, better (24 of 67 [35.8%]). Organisms on the WT-stained smear exhibited better morphology in a few instances (4 of 67 [6%]), and in three instances, there were discrepant organism numbers.     

Suppression of in vitro growth of virulent and avirulent herpes simplex viruses by cell-mediated immune mechanisms, antibody, and interferon.

A rounding cell-forming--GC strain, which is a variant of a syncytial giant cell-forming herpes simplex virus (+GC Miyama strain), was highly attenuated for Swiss, BALB/c nu/nu, and nu/+ mice, whereas +GC was highly virulent to all the mice tested. +GC and -GC were antigenically indistinguishable from each other by cross-neutralization and cross-immunization. Immunosuppression induced by cyclophosphamide converted the nonlethal -GC infection of mice into a fatal infection. -GC replication in tissue culture was more effectively suppressed by spleen cells immunized with either +GC or -GC than was the +GC replication. -GC replication was also inhibited more effectively by antibody or the antibody-dependent cell-mediated system than was the +GC replication. -GC is highly sensitive to mouse interferon, but +GC was relatively resistant. These findings indicate that attenuation of this avirulent -GC strain may be due to a high susceptibility of its replication to humoral and cell-mediated defense factors. The probable roles of each defense factor in recovery from the infection with virulent and attenuated herpes simplex virus are also discussed.     

Roles of tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), and IL-10 in the modulation of intercellular adhesion molecule-1 (ICAM-1) expression by macrophages during mycobacterial infection

Profiles of ICAM-1 expression on cultured murine peritoneal macrophages infected with Mycobacterium avium complex (MAC) were examined, with special reference to modulating roles of TNF-alpha, TGF-beta, and IL-10. When macrophages were infected with MAC, ICAM-1 expression, measured by microscopic counting of ICAM-1+ macrophages stained with anti-ICAM-1 antibody, ELISA, and flow cytometric analysis, was rapidly increased, peaking at day 3 (early-phase up-regulation) due to endogenous TNF-alpha, and thereafter gradually declined to the normal level within 1 week or more (late-phase down-regulation). The late-phase ICAM-1 down-regulation was also seen in macrophages phagocytosing heat-killed MAC and those stimulated with lipopolysaccharide but not in macrophages phagocytosing latex beads. ICAM-1 mRNA expression was augmented markedly at day 1 after MAC infection and thereafter decreased. While TNF-alpha and IL-10 production by MAC-infected macrophages was observed during the first 3 days, TGF-beta production was initiated from day 3 and continued until day 14. Exogenously added TGF-beta strongly inhibited the early-phase increase in ICAM-1 expression by infected macrophages, and the blockade of endogenous TGF-beta with anti-TGF-beta antibody markedly inhibited late-phase ICAM-1 down-regulation. Moderate blocking effect was also observed for anti-IL-10 antibody. On the other hand, late-phase ICAM-1 down-regulation was not prevented by the addition of exogenous TNF-alpha. Therefore, TGF-beta and IL-10, especially the former, appear to play active roles in the late-phase down-regulation of ICAM-1 in MAC-infected macrophages during long-term cultivation.     

Priming of the oxidative burst in human neutrophils by physiological agonists or cytochalasin B results from the recruitment of previously non-responsive cells.

Using a sensitive flow cytometric assay, which measures the intracellular oxidation of 2'7' dichlorofluorescein (DCFH) by H2O2, we have assessed, at a single-cell level, the effects of a variety of physiological priming agonists and cytochalasin B (CB) on purified populations of neutrophils stimulated at different points along the signal response transduction pathway. Pretreatment of purified neutrophils with the physiological priming agonists monocyte interleukin-8 (IL-8), granulocyte-monocyte colony-stimulating factor (GM-CSF), platelet-activating factor (PAF), IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), IL-6, and non-stimulatory doses of formyl-methionyl-leucyl-phenylalanine (FMLP), resulted in an increased percentage of cells generating an oxidative burst in response to subsequent receptor stimulation with FMLP. CB had a similar but much more pronounced effect on cellular recruitment to a receptor-mediated responsive state. Activation of protein kinase C (PKC) using the phorbol ester phorbol myristate acetate (PMA) resulted in a heterogeneous response, with all cells generating H2O2, but with two populations differing in their magnitude of response. Physiological priming agonists had no effect on the heterogeneity of the PMA response. However, pretreatment with CB dramatically altered the PMA response, producing a homogeneous population highly responsive to stimulation with PKC. In contrast, direct stimulation of G proteins with fluoride (A1F-4) was primed both by physiological priming agonists and by CB. These results demonstrate that priming of neutrophils by physiological agonists involves changes at the level of signal transduction which enable a previously non-responsive cell to respond to a secondary stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of intestinal parasite morphology in polyvinyl alcohol preservative: comparison of copper sulfate and mercuric chloride bases for use in Schaudinn fixative.

As a result of disposal problems inherent in the use of mercury compounds, many laboratories have considered using copper sulfate as a substitute for mercuric chloride in polyvinyl alcohol (PVA) preservative. The primary use for PVA-preserved specimens is the permanent stained smear, the most important technique for the identification of intestinal protozoa. A comparison of organism recovery and morphology was undertaken with PVA containing either copper sulfate or mercuric chloride base. Paired fecal specimens (417 pairs) were collected and examined with the Formalin-ether concentration and Trichrome stain techniques. Numbers of organisms recovered and helminth egg and protozoan morphology were assessed from the concentration sediment. Morphology, clarity of nuclear and cytoplasmic detail, overall color differences, and the ease or difficulty in detecting organisms in fecal debris were assessed from the permanent stained smear. No significant differences were found in the numbers and morphology of organisms seen in the concentration sediment. However, when the trichrome stain was used, the overall morphology of the intestinal protozoa preserved in PVA with copper sulfate was not equal to that seen with PVA with mercuric chloride. We do not recommend switching from mercuric chloride base to copper sulfate base unless that is the only option available for the preparation of permanent stained smears.