Measuring interoception: The phase adjustment task.

Interoception, perception of one's bodily state, has been associated with mental health and socio-emotional processes. However, several interoception tasks are of questionable validity, meaning associations between interoception and other variables require confirmation with new measures. Here we describe the novel, smartphone-based Phase Adjustment Task (PAT). Tones are presented at the participant's heart rate, but out of phase with heartbeats. Participants adjust the phase relationship between tones and heartbeats until they are synchronous. Data from 124 participants indicates variance in performance across participants which is not affected by physiological or strategic confounds. Associations between interoception and anxiety, depression and stress were not significant. Weak associations between interoception and mental health variables may be a consequence of testing a non-clinical sample. A second study revealed PAT performance to be moderately stable over one week, consistent with state effects on interoception.

Chromosomal location of a human alpha interferon gene family.

To determine the chromosomal location of the human alpha interferon genes, we scored a series of human/rodent somatic cell hybrids for the presence of DNA sequences hybridizing to an alpha 1 interferon DNA probe. The presence of human chromosome 9 in a hybrid correlated with the presence of a family of alpha interferon genes.

A GPI-anchored co-receptor for tissue factor pathway inhibitor controls its intracellular trafficking and cell surface expression.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) lacks a membrane attachment signal but it remains associated with the endothelial surface via its association with an, as yet, unidentified glycosyl phosphatidylinositol (GPI)-anchored co-receptor. OBJECTIVES/METHODS: Cellular trafficking of TFPI within aerolysin-resistant ECV304 and EA.hy926 cells, which do not express GPI-anchored proteins on their surface, was compared with their wild-type counterparts. RESULTS AND CONCLUSIONS: Although aerolysin-resistant cells produce normal amounts of TFPI mRNA, TFPI is not expressed on the cell surface and total cellular TFPI is greatly decreased compared with wild-type cells. Additionally, normal, not increased, amounts of TFPI are secreted into conditioned media indicating that TFPI is degraded within the aerolysin-resistant cells. Confocal microscopy and studies using metabolic inhibitors demonstrate that aerolysin-resistant cells produce TFPI and transport it into the Golgi with subsequent degradation in lysosomes. The experimental results provide no evidence that cell surface TFPI originates from secreted TFPI that binds back to a GPI-anchored protein. Instead, the data suggest that TFPI tightly, but reversibly, binds to a GPI anchored co-receptor in the ER/Golgi. The co-receptor then acts as a molecular chaperone for TFPI by trafficking it to the cell surface of wild-type cells or to lysosomes of aerolysin-resistant cells. TFPI that escapes co-receptor binding is secreted through the same pathway in both wild-type and aerolysin-resistant cells. The data provide a framework for understanding how TFPI is expressed on endothelium.

Intragraft proliferating T lymphocytes are associated with moderate acute pulmonary rejection.

Acute allograft rejection is characterized by infiltration of the donor organ by host lymphoid cells, predominantly T lymphocytes. However, the site of proliferation and clonal expansion of alloreactive T lymphocytes is not well defined in man. A group of normal transbronchial biopsies (TBB, n=9) from clinically well lung transplant recipients was compared to TBB showing acute rejection (at least grade A2, n=9), using CD3- and Ki67-specific antibodies to double-label proliferating T lymphocytes. Few double-labeled lymphocytes were present in the normal biopsies (range, 0-3 cells). However, five of the rejection biopsies contained significant numbers of proliferating T lymphocytes (range, 19-47; Fisher's exact test; P=0.029). Furthermore, this positive group contained all three cases of grade A3 rejection in the study, as well as a case with persistent grade A2 rejection on follow-up biopsy. These data demonstrate that T lymphocytes do proliferate in transplanted human lungs; such proliferation is associated with more severe rejection.

UK-78,282, a novel piperidine compound that potently blocks the Kv1.3 voltage-gated potassium channel and inhibits human T cell activation.

1. UK-78,282, a novel piperidine blocker of the T lymphocyte voltage-gated K+ channel, Kv1.3, was discovered by screening a large compound file using a high-throughput 86Rb efflux assay. This compound blocks Kv1.3 with a IC50 of approximately 200 nM and 1:1 stoichiometry. A closely related compound, CP-190,325, containing a benzyl moiety in place of the benzhydryl in UK-78,282, is significantly less potent. 2 Three lines of evidence indicate that UK-78,282 inhibits Kv1.3 in a use-dependent manner by preferentially blocking and binding to the C-type inactivated state of the channel. Increasing the fraction of inactivated channels by holding the membrane potential at - 50 mV enhances the channel's sensitivity to UK-78,282. Decreasing the number of inactivated channels by exposure to approximately 160 mM external K+ decreases the sensitivity to UK-78,282. Mutations that alter the rate of C-type inactivation also change the channel's sensitivity to UK-78,282 and there is a direct correlation between tau(h) and IC50 values. 3. Competition experiments suggest that UK-78,282 binds to residues at the inner surface of the channel overlapping the site of action of verapamil. Internal tetraethylammonium and external charybdotoxin do not compete UK-78,282's action on the channel. 4. UK-78,282 displays marked selectivity for Kv1.3 over several other closely related K+ channels, the only exception being the rapidly inactivating voltage-gated K+ channel, Kv1.4. 5. UK-78,282 effectively suppresses human T-lymphocyte activation.

An in vitro system to model pulmonary epithelial barrier dysfunction mediated by immune effector cells.

An assay of epithelial barrier function was developed to monitor immune-mediated changes in lung permeability that may be occurring during pulmonary allograft rejection and inflammatory lung diseases. Lung tissue was obtained from minipigs, digested with collagenase (1 mg/ml) overnight, and propagated in RPMI 1640 tissue culture medium. Cells with an epithelioid morphology were purified by differential detachment using trypsin-ethylenediaminetetraacetic acid and were characterized as epithelial by positive staining with an anti-cytokeratin monoclonal antibody. Monolayers of these epithelial cells were cultured on porous tissue culture inserts, and transmonolayer resistance values were measured. Transmonolayer resistance values reached a mean of 5487 +/- 2882 omega (mean +/- 95% confidence interval; n = 9) after 5 days in culture. These values indicated the presence of functional intercellular tight junctions between the cells. Addition of cytotoxic immune effector cells to the cultured monolayers caused a rapid reduction in the transmonolayer resistance values, whereas unstimulated splenocytes failed to produce this effect. Comparison of these results with those obtained in parallel experiments performed with standard isotopic cytotoxicity assays indicated the sensitivity of the transmonolayer resistance technique. The assay described in this report will enable in vitro modeling of epithelial permeability damage mediated by both activated lymphoid cells and their soluble products.

Limiting dilution analysis: quantification of IL-2 producing allospecific lymphocytes after renal and cardiac transplantation.

A rapid and robust limiting dilution assay was developed to measure the frequency of donor-reactive, IL-2 (interleukin 2) producing, helper T lymphocytes in the peripheral T cell population of organ allograft recipients. The IL-2 bioassay was performed using two methodologies to assess the response of CTLL-2 indicator cells. The first depended on spectrophotometric detection of bioreduced XTT whilst the second involved measurement of [3H]thymidine incorporation. The radioisotopic method was slightly more sensitive but both assays could be used for analysis of limiting dilution culture supernatants after primary incubation of recipient lymphocytes with donor splenic cells for 48 hours. All the assays produced results which conformed to single hit kinetics, indicating that IL-2 was production was dependent on a single limiting cell type. The frequency of allospecific helper lymphocytes in the peripheral T cell population of normal volunteers did not vary significantly during a 28-day period. It was found that immunosuppressed allograft recipients had a significantly reduced proportion of T cells in their peripheral blood mononuclear cell population. However, it was possible to measure the frequency of donor-reactive helper cells in the T cell population of transplant patients. These frequency values were very low in two renal allograft recipients who were HLA-DR matched to their donor organs. Three of four HLA-DR mismatched cardiac recipients showed a significant decrease in the frequency of their donor-reactive helper lymphocytes during the period of monitoring. The fourth patient, who received antilymphocyte antibodies for the first three days after transplantation, showed significant fluctuations in the frequency of these cells. The four cardiac recipients showed little histopathological evidence of acute graft rejection with only one patient experiencing a brief episode of moderate rejection; this patient showed a high frequency of donor-reactive helper cells when assayed immediately after this episode but the frequency subsequently declined.

Specific, high affinity colchicine binding monoclonal antibodies: development and characterization of the antibodies.

Nine colchicine specific monoclonal antibodies have been developed by immunizing BALB/c mice with a colchicine-keyhole limpet hemocyanin (Col-KLH) conjugate prepared using a bishydroxysuccinimide coupling reagent. Of four immunization procedures examined, intraperitoneal injection of the antigen attached to acid treated E. coli resulted in the maximum antigen specific antibody titers. A colchicine bovine serum albumin (Col-BSA) conjugate, prepared using a water soluble carbodiimide coupling technique, formed the basis of an enzyme linked immunosorbent assay used for screening hybridomas for colchicine specific antibody secretion and for determining the relative affinity and specificity profile of the monoclonal antibodies. All antibodies demonstrated high affinity, saturable binding to colchicine and low cross-reactivity with a panel of compounds structurally related to colchicine. The IC50 for the highest affinity antibody, C44, was 3.6 +/- 0.84 nM colchicine in the competitive enzyme immunoassay. The affinity of this antibody determined from Scatchard analysis of antibody binding to tritiated colchicine was 0.66 +/- 0.11 nM. Antibody C44 has the level of specificity and affinity suitable for a sensitive and selective immunoassay of colchicine for monitoring therapeutic drug levels. In addition, this antibody provides a specific pharmacologic antagonist for studies of colchicine's therapeutic mechanism and has the potential to reverse colchicine toxicity.

Role of the mucosal integrin alpha(E)(CD103)beta(7) in tissue-restricted cytotoxicity.

The effectiveness of lung transplantation is marred by the relatively high incidence of rejection. The lung normally contains a large population of lymphocytes in contact with the airway epithelium, a proportion of which expresses the mucosal integrin, alpha(E)(CD103)beta(7). This integrin is not a homing receptor, but is thought to retain lymphocytes at the epithelial surface. Following transplantation, a population of 'tissue-restricted' cytotoxic T cells (CTL) have been identified which have the ability to lyse epithelial cells, but not major histocompatibility complex (MHC)-identical splenic cells. We tested the hypothesis that expression of the mucosal integrin confers the ability of CTL to target and destroy e-cadherin expressing targets. Immunohistochemical and flow cytometric analyses were used to demonstrate the relevance of this model to human lung. Allo-activated CTL were generated in mixed leucocyte reactions and CD103 expression up-regulated by the addition of transforming growth factor (TGF)-beta. The functional effect of CD103 expression was investigated in (51)Cr-release assays using e-cadherin-expressing transfectant targets. Human lung epithelial cells express e-cadherin and one-third of intraepithelial lymphocytes (IEL) expressed CD103. Allo-activated and bronchoalveolar lavage (BAL) lymphocytes express more CD103 than those in blood. Transfection of e-cadherin into murine fibroblasts conferred susceptibility to lysis by alpha(E)beta(7)-expressing CTL which could be blocked by specific monoclonal antibodies to CD103 and e-cadherin. CD103 functions to conjugate CTL effectors to e-cadherin-expressing targets and thereby facilitates cellular cytotoxicity. E-cadherin is expressed prominently by epithelial cells in the lung, enabling CTL to target them for destruction.

Regulation and function of adhesion molecule expression by human alveolar epithelial cells.

The role of major histocompatibility complex (MHC) and adhesion molecule expression by alveolar epithelium on the modulation of immune responses in the lung is not understood. We have developed efficient methods to isolate, purify and culture human alveolar epithelial cells (type II pneumocytes) in vitro. The expression of MHC and adhesion molecules by isolated, cultured and cytokine-stimulated alveolar epithelial cells was quantified by flow cytometry, and demonstrated the presence of T-cell ligands including class I MHC, HLA-DR and HLA-DP, intracellular adhesion molecule-1 (ICAM-1; CD54) and lymphocyte function-associated antigen (LFA-3; CD58), but not vascular cell adhesion molecule-1 (VCAM-1) (CD106) or B7 (CD80). The proinflammatory cytokine interferon-gamma (IFN-gamma) caused an up-regulation of class I MHC and ICAM-1. In contrast, tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) had little effect on the expression of these surface antigens by human alveolar epithelial cells. The functional activity of alveolar epithelial adhesion molecules was then studied by determining their ability to bind allogeneic lymphocytes. An increase in lymphocyte adherence to monolayers of alveolar epithelial cells was observed following in vitro activation. However, up-regulation of alveolar epithelial counter receptors with the proinflammatory cytokine gamma-IFN did not enhance adhesion. The adhesive interaction between CD18 on lymphocytes and ICAM-1 on alveolar epithelial cells was demonstrated by the use of blocking antibodies specific for both ligands. Blockade of LFA-3 on alveolar monolayers also suppressed lymphocyte adherence. In conclusion, alveolar epithelial cells expressed MHC HLA-A, B, C, HLA-DR and -DP, and functional adhesion molecules including ICAM-1 and LFA-3.

Alloantigen presentation by B cells: analysis of the requirement for B-cell activation.

This paper describes a model for investigation of the functional implications of B-cell activation for antigen presentation. Mixed lymphocyte cultures were used to assess the ability of freshly isolated B cells, mitogen-activated B cells and Epstein-Barr virus (EBV)-transformed B-cell lines to stimulate the activation and proliferation of allogeneic T cells under a variety of experimental conditions. It was found that resting B cells presented antigen poorly, while activated cells were highly immunogenic. Paraformaldehyde fixation completely eliminated antigen presentation by resting B cells, despite constitutive expression of class II MHC antigens. However, fixation had little effect on antigen presentation by activated B cells that expressed B7-1 and B7-2 in addition to class II major histocompatibility complex (MHC) molecules. Arrest of B-cell activation by serial fixation after treatment with F(ab')2 fragments of goat anti-human IgM produced cells with variable antigen-presenting capacity. Optimal antigen presentation was observed for cells fixed 72 hr after the initiation of B-cell activation. Although both B7-1 and B7-2 antigen expression increased after B-cell activation, it was found that the rate of T-cell proliferation correlated most closely with B7-2 expression. Stimulation of T cells by fixed activated B lymphocytes could be blocked by antibodies directed at class II MHC molecules, indicating involvement of the T-cell antigen receptor. In addition, T-cell proliferation was inhibited by antibodies specific for B7-1 and B7-2 and by the fusion protein CTLA4-Ig, demonstrating a requirement for CD28 signal transduction. The sole requirement of B7 family expression for antigen presentation by B lymphocytes was shown by demonstration of T-cell stimulation by fixed resting B cells in the presence of CD28 antibody as a source of artificial costimulation.

Demonstration of direct xenorecognition of porcine cells by human cytotoxic T lymphocytes.

It is not known whether human cytotoxic T cells can recognize porcine major histocompatibility antigens directly, or whether recognition occurs by co-operation with syngeneic human antigen-presenting cells (APC). Limiting dilution assays were used to quantify human anti-pig precursor cytotoxic T-cell (CTLp) frequencies and to analyse the 'kinetics' of the interaction between human lymphoid cells and porcine splenic cells. Single-hit kinetics are demonstrative of direct recognition, as only one cell type, the CTLp, is diluted out, whereas multi-hit kinetics indicate that more than one cell is limiting and provide evidence for co-operative recognition of xenoantigens. Initial assays indicated that the frequency of CTLp reactive with alloantigens on human splenic targets (mean 1/1845; n = 3) was approximately sixfold greater than the frequency of CTLp reactive with porcine splenic cells (1/12,082; n = 3). However, not all of the assays performed using the xenogeneic combination produced single-hit kinetics. Subsequent assays were performed by mixing limiting numbers of human peripheral blood mononuclear cells (PBMC) or APC-depleted PBMC preparations with porcine splenocytes. There was a significant difference in the frequency of xenospecific CTLp between PBMC and APC-depleted preparations (P = 0.034). The overall frequency increased in the APC-depleted group. Variation between the seven human donors was also significant (P = 0.006). There was no significant difference in frequency between the two cell preparations after correction for the proportion of CD3+ cells (P = 0.13). There was, however, a significant departure from single-hit kinetics in the PBMC group (P = 0.004) which was not observed in the APC-depleted group (P = 0.052). It is concluded that human cytotoxic T cells can be activated by porcine xenoantigens directly. However, the direct recognition mechanism can be altered in the presence of human APC.

Primary human alveolar epithelial cells can elicit the transendothelial migration of CD14+ monocytes and CD3+ lymphocytes.

The ability of freshly isolated primary human alveolar epithelial cells (type II pneumocytes) to induce leucocyte migration across an endothelial monolayer was investigated. Three-way factorial analysis of variance (ANOVA) demonstrated that resting alveolar endothelial cells (AEC) could produce detectable quantities of monocyte chemoattractant protein 1 (MCP-1), which was upregulated in response to tumour necrosis factor-alpha (TNF-alpha) in a dose- and time-dependent fashion. Interferon-gamma (IFN-gamma) had no significant effect on this process. TNF-alpha and IFN-gamma both induced AEC to provoke migration of CD14+ monocytes and CD3+ lymphocytes across endothelium. IFN-gamma and TNF-alpha synergized in their ability to induce production of T lymphocyte, but not monocyte, chemoattractants from AEC. Leucocyte transendothelial migration was inhibited by anti-MCP-1 neutralizing antibody and by heparin, a polyanionic glycosaminoglycan (GAG). These data suggest that human AEC play a role in the multiple mechanisms that facilitate monocyte and T lymphocyte migration into the alveolar compartment of the lung under homeostasis and inflammatory conditions. One of these mechanisms is mediated via constitutive MCP-1 production by alveolar epithelial cells, which is upregulated by TNF-alpha.

A pharmacologic study of the relationship between lymphocyte function and surface antigen expression.

The relationship between functional activity and distribution of lymphocyte surface markers has not been clearly defined. We have examined the relationship between cell surface markers and function under the influence of immunosuppressant therapy. We found that after immunization with EL4 cells, the development of the immune response in the BALB/c mouse was accompanied by a decrease in spleen cells which stained brightly with fluorescein-labeled monoclonal anti-Thy 1 and an increase in cells which stained with rabbit anti-mouse Ig as measured on the FACS. Low doses of azathioprine and cyclophosphamide, which affect functional activity of the cells, do not alter cell surface markers. However, at higher doses of the drugs normalization of immunization-induced marker changes were observed, and the Thy 1+ and Ig+ surface markers were maintained at levels seen in non-immunized mice. In spite of a nearly 3-fold increase in the total number of lymphocytes and an increase in the functional activity of cytotoxic T cells (Lyt 2+) after immunization, no alteration in the percentage of Lyt 2+T cells nor in the intensity of staining with FITC-labeled Lyt2 antibody was seen. Inhibition of the immune response with immunosuppressant also failed to change the Lyt2+-staining cell population. This study demonstrates that lymphocyte functional changes precede cell surface antigen changes, and that functional changes may occur without surface antigen changes. Thus cell surface markers are "late" indicators of functional changes.

Humoral responses to Malassezia furfur serovars A, B and C in normal individuals of various ages.

A transferable solid-phase (TSP) ELISA was developed for the determination of antibody titres specific to Malassezia furfur serovars A, B and C in human sera. A survey of levels of class-specific antibodies (IgM, IgG and IgA) to M. furfur serovars A, B and C in relation to age (2-64 years; 60 individuals) demonstrated that individuals had immunity to M. furfur by the age of 2-3 years. There was no difference in either IgM or IgG levels into adulthood. The only age-related differences were lower IgM titres to the three serovars in the 60-64 year age-group compared with younger individuals. There was, however, a difference between titres of antibody specific to the three serovars. The mean reciprocal log2 IgM titre to serovar A (6.9) was significantly higher (P < 0.05) than that to serovar B (mean reciprocal log2 titre of 5.8), but not to serovar C (6.1). In contrast, the mean reciprocal log2 IgG titre to serovar A (6.5) was significantly lower (P < 0.05) than those to serovars B and C (mean reciprocal log2 titre of 8.9 in both cases).

Functional restoration for chronic low back pain: Changes in depression, cognitive distortion, and disability.

In the present study, 107 patients (72 males and 35 females) completed self-report measures of depression, distortion, disability, and pain intensity at three points during their rehabilitation: (1) admission to a 3-week comprehensive functional restoration program, (2) discharge from the comprehensive phase, and (3) 4-6 weeks later at their first post-program evaluation. Various range-of-motion measures were also collected at these same times using inclinometry. Results demonstrated significant improvements on all measures which were maintained into follow-up. Patients were also subsequently grouped into depressed and non-depressed at admission, and both groups demonstrated significant improvement across time. Additionally, patients were divided into high and low distortion groups. High general cognitive distortion patients did not show improvement on 3 of the 5 range of motion, or pain intensity scores, although they did improve on their depression, distortion, and disability scores. Findings also suggested thatlow back pain-related cognitive distortion may be considered a state or situational factor, whereasgeneral cognitive distortion appears to be more of a trait characteristic.

The gene coding the human S11 surface antigens maps between the loci for HPRT and G6PD on the X-chromosome.

The human S11 surface antigens are expressed on fibroblasts and are coded by a gene on the X-chromosome. We have regionally mapped this gene by examining S11 expression on a panel of hybrid lines which had fragmented the X-chromosome either during chromosome-mediated gene transfer, or by interspecies translocation during hybrid cell expansion. using indirect immunofluorescence and the fluorescence-activated cell sorter (FACS), it was possible to isolate antigen-positive and -negative hybrid subpopulations for subsequent genetic analysis. The gene coding S11 could be localized to Xq27-28, between the loci for HPRT and G6PD where genes for the S10 and S12 antigens have been previously mapped. This work demonstrates the value of cell surface antigens and the FACS in somatic cell genetic analysis, and provides evidence for regional clustering of surface antigen loci on the human X-chromosome.

A comparison of the antigen-presenting capabilities of class II MHC-expressing human lung epithelial and endothelial cells.

Human lung alveolar epithelial cells constitutively express class II major histocompatibility complex (MHC). Human lung microvascular endothelial and small airway epithelial cells can be induced to express class II MHC by stimulation with the pro-inflammatory cytokine interferon-gamma. The levels of class II MHC on lung epithelial and endothelial cells were comparable to those seen on an Epstein-Barr virus (EBV)-transformed B-cell line. However, the costimulatory molecules B7-1 and B7-2 were not expressed. The ability of the class II MHC expressing human lung parenchymal cells to present alloantigen to CD4+ T lymphocytes was investigated. Freshly isolated human alveolar epithelial cells (type II pneumocytes) and monolayers of interferon-gamma-stimulated small airway epithelial and lung microvascular endothelial cells were co-cultured with allogeneic CD4+ T lymphocytes and proliferation determined by [3H]thymidine incorporation. A clear difference was observed between effects of the epithelial and endothelial cells on CD4+ T-lymphocyte activation. Alveolar and small airway epithelial cells failed to stimulate the proliferation of allogeneic CD4+ T lymphocytes whereas lung microvascular endothelial cells did stimulate proliferation. This difference could not be explained by the levels of class II MHC or the lack of B7-1 and B7-2 solely. Microvascular endothelial cells, and not alveolar or small airway epithelial cells, possess B7-independent costimulatory pathways.

Cyclophosphamide and 15(S)-15 methyl PGE1 correct the T/B lymphocyte ratios of NZB/NZW mice.

The lupus of NZB/NZW F1 female mice is associated with immune complex glomerulonephritis and premature death. Cyclophosphamide and 15(S)-15 methyl PGE1 therapy halt disease progression. Fluorescein conjugated antibodies were utilized to label specific leukocytes and the subsets were quantitated using a Fluorescence Activated Cell Sorter. Normal outbred CD-1 female mice showed a decrease in absolute T and B cell numbers with age, but the ratio of T and B cells remained essentially constant through 9 months of age. By contrast the NZB/W female mice showed decreased numbers of total lymphocytes relative to CD-1 controls at all ages. Moreover relative to CD-1s, there was a far greater decrease in T cell numbers (7 x for NZB/W versus 2 x for CD-1) and B cell numbers failed to decrease with age. The characteristic decline in T lymphocyte numbers and relative increase in B cell numbers in NZB/W mice were corrected with cyclophosphamide and PGE1 therapy. However, there was no selective modification of T cell subsets (L3T4+ or Ly2+) with therapy. Our investigation suggests correction of the abnormal T/B cell ratio may be a useful marker of therapeutic activity in NZB/W mice.

Differentiation of three serovars of Malassezia furfur.

Malassezia furfur strains were isolated from the clinically normal skin of 10 volunteers by swabbing four different sites (forehead, ear, back and chest). The strains could be divided into three basic groups on the basis of cultural characteristics. Both unabsorbed and absorbed specific rabbit antisera were prepared against nine of the strains, and both species and group specific antigens could be demonstrated. Serologically, three group specific surface antigens could be identified which corresponded to the three groups identifiable on cultural characteristics. The relevance of these findings to previous in vitro results is discussed.