That dog won't fit: body size awareness in dogs.

With very few exceptions, no coherent model of representing the self exists for nonhuman species. According to our hypothesis, understanding of the Self as an object' can also be found in a wide range of animals including the dog, a fast-moving terrestrial predator/scavenger, with highly developed senses and complex cognitive capacity. We tested companion dogs in three experiments in which they faced three different variations of the same physical challenge: passing through an opening in a wall. We predicted that if dogs are capable of representing their own body size, they will react differently when faced with adequate or too small openings. We found that dogs started to move towards and approached the too small openings with significantly longer latencies than the suitable ones; and upon reaching it, they did not try to get through the too small openings. In another experiment, the medium-size (still large enough) opening was approached with latencies that fell between the latencies measured in the cases of the very large or the too small openings. Having discussed the potential underlying mechanisms, we concluded that our results convincingly assume that dogs can represent their own body size in novel contexts.

An immunological hypothesis for plasma protein catabolism.

A selective mechanism of plasma protein catabolism is suggested, based on three main assumptions: (1) plasma proteins are submitted to molecular ageing thus achieving "modified" forms after a given time period; (2) the system recognizing the "modified" molecules consists of preformed physiological autoantibodies; (3) the elimination of the immune complexes formed between "modified" proteins and the corresponding autoantibodies takes place via binding to Fc receptor bearing cells.

Determination of the antibody binding capacity of lymphocyte membrane antigens by flow cytometry in 58 blood donors.

The relative density of lymphocyte CD3, CD4, CD5, CD8, CD20, CD23, CD28, CD38, CD45RA, CD45RO, CD57 and HLA-DR antigens was measured as antibody binding capacity (ABC) in 58 blood donors aged 19-66 years. The group was analysed in order to obtain reference values (percentages and absolute numbers) for routine, quantitative three-colour flow cytometry (FC) tests, and we included around ten males and females for each of the 15 year age intervals. Whole blood was stained (30 min on ice) with FITC, PE or PerCP conjugated MAbs. The analysis was performed with a FACScan equipped with LYSYS II and Paint-a-GatePlus software. The instrument was calibrated daily with QC3, QuickCal (FITC and PE) and Calibrite and monthly with QSC and stained cells (which included also the control for PerCP performance). The ABC was measured with QSC (Flow Cytometry Standards Corporation). The CD4+ lymphocytes expressed significantly more CD3, CD28 and HLA-DR antigens, and less CD45RA antigen than the CD8+ cells (p < 0.0001). A significant decrease with age was observed for CD3 and CD45RA on both CD4+ and CD8+ subsets (p < 0.05). The lymphocytes of women, compared with those of men, showed decreased ABC for CD8, CD20 and CD28 antigens. The results illustrate the necessity for close matching of control with case groups. They also illustrate the possibilities of modern FC methods based on quantitative quality control and three-colour analysis.

Methods for detection of anti-albumin autoantibodies in hepatic diseases.

Three methods for detection and quantitation of anti-albumin autoantibodies (AAA) in human sera have been developed based on their reaction with a new antigenic site of glutaraldehyde-polymerized human serum albumin (HSA). The first is a double diffusion technique (ID-HSA) were the sera to be tested are reacted with a copolymer of glutaraldehyde activated HSA (copoly-HSA). The reaction is estimated by the lowest copoly HSA concentration giving precipitation with the undiluted sera. The second method is based on agglutination (HA) by AAA positive sera of human red blood cells coated with glutaraldehyde-polymerized HSA (EPA). The reaction is quantified as the highest dilution of sera giving agglutination. The third method uses glutaraldehyde insolubilized HSA as an immunoadorbent able to bind AAA in positive sera. The quantity of protein adsorbed, expessed in microng antibody/ml serum, measures the level of AAA in sera. Significant correlation between the values of AAA as determined by all three methods presented above were recorded.

Reactivity of sera from patients with liver disease with glutaraldehyde-treated erythrocytes: role for false positive results in hemagglutination tests.

The use of glutaraldehyde as a coupling reagent in the passive hemagglutination test (HA) has gained wide application, especially for the coating of red blood cells (RBC) with glutaraldehyde-polymerized human serum albumin (pHSA), for studies of the albumin receptor on hepatitis B surface antigen (HBsAg) or for the detection of anti-albumin antibodies (AAA). Here we report a previously unrecognized reactivity with glutaraldehyde-treated RBC mainly with sera from patients with liver disease. The highest incidences of this reaction were found in patients with acute viral hepatitis A and B, namely 44 of 50 (88%) and 31 of 50 (62%) respectively. In 234 HBsAg carriers the frequency was low (3%). This reactivity was also observed in 19 of 50 sera from patients with chronic liver disease documented by biopsy, but not in sera from 68 healthy subjects. By immunofluorescence on glutaraldehyde-treated RBC it was shown that the corresponding antibodies belonged mainly to the IgM class. In all HBsAg-negative patients studied the HA titer against glutaraldehyde-treated RBC was in agreement with the titer against RBC coated with pHSA or pBSA (polymerized bovine serum albumin). Absorption with pHSA abolished the reaction with glutaraldehyde-treated RBC in 7 of 8 sera, suggesting a common reactivity between glutaraldehyde-polymerized HSA and glutaraldehyde-treated RBC. Apart from the possible clinical importance of these antibodies, their existence is a possible source of false positive results when glutaraldehyde is used as a coupling reagent for immunological assays, in particular with sera from patients with liver diseases.

Clinical and immunological benefits from highly active antiretroviral therapy in spite of limited viral load reduction in HIV type 1 infection.

Both naive and memory T lymphocyte responses are lost during advanced HIV infection. Treatment with highly active antiretroviral therapy (HAART) is associated with an increase in T lymphocytes and a reduction in viral load. However, the viral response to HAART in patients with low levels of helper T lymphocytes and a high viral load is often not satisfactory. We investigated the capacity of long-term HAART to reconstitute the immune system in severely ill patients. A nonselected longitudinal patient population with high baseline viral levels and CD4(+) cells below 100 x 10(6)/liter were monitored for 2 years during HAART. Markers to estimate the therapeutic effects included viral levels and cell surface markers representing naive and memory T lymphocytes as well as activation markers, B cells, NK cells, and clinical events. After 2 years of treatment, viral load was reduced to undetectable levels in 55% (viral responders, vRs) and less than 1 log (median value) from baseline in 45% (viral low responders, vLRs). Elevated numbers of memory and naive CD4(+) and CD8(+) cells as well as a decrease in activation markers were seen in both vRs and vLRs. However, the magnitude was greater in vRs. No differences in the clinical outcome were observed between vRs and vLRs. We conclude that most patients, even in advanced stages of HIV disease, benefited from HAART. The magnitude of the response was related to good viral reduction, but even patients with poor viral reduction had a recovery of naive and memory CD4(+) and CD8(+) cells. Even a small reduction in viral load is thus of importance for health and potentially also for years of survival.

DNA immunization with HIV early genes in HIV type 1-infected patients on highly active antiretroviral therapy.

The aim of this study was to evaluate the immunological responses induced by DNA plasmids containing HIV regulatory genes administered in combination in HIV-1-infected patients with pretreatment with highly active antiretroviral treatment (HAART). The study is a double-blind, randomized, and placebo-controlled study, including 15 asymptomatic HIV-1-infected patients on stable HAART for at least 6 months and with plasma HIV RNA levels below 50 copies/ml. Ten patients received a combination of rev, tat, and nef intramuscularly (im) at weeks 0, 4, and 16 at increasing doses giving totals of 300 (100 x 3), 900 (300 x 3), and 1800 (600 x 3) micrograms DNA. Five patients received saline in the same amounts im. Antigen-specific cytotoxic T lymphocyte (CTL) levels were preserved or increased and new T lymphocyte proliferative responses were induced in the group immunized with the HIV DNA genes. No increase in antibody levels was noted. Despite a 10-fold higher vaccine dose, patients on HAART did not respond better to vaccination compared to non-HAART patients included in a previous study where the genes were administered separately. Combining the regulatory genes rev, tat, and nef in increasing doses may reduce the anticipated augmentation of HIV-specific T cell proliferative and CTL responses. Viral suppression did not seem to further improve the initial vaccine responses of patients with comparable CD4 levels.

Expression of CD15 as predictor of relapse in children with acute lymphoblastic leukemia of the pre-B type.

Pre-B acute lymphoblastic leukemia (ALL) was diagnosed in 37 children morphologically, histochemically, and by immunophenotyping by flow cytometry. In all patients the leukemic blasts expressed HLA-DR and CD19 (Leu-12). In 10 patients 20% or more of the blast cells expressed a myeloid antigen: CD15 (Leu-M1) in seven, CD33 (My9) in two and CD13 (My7) in one patient. All 37 children achieved complete remission, but eight relapsed. Relapse occurred in six of seven patients with CD15-positive blasts, but in only two of 27 patients with CD15-negative blasts (p = 0.0003). Thus, the occurrence of CD15 on the blasts of children with pre-B ALL shows a remarkable association with a high risk of relapse, and these patients should therefore be considered to belong to the high-risk group regardless of other prognostic factors.

Variability in B-cell antigen expression: implications for the treatment of B-cell lymphomas and leukemias with monoclonal antibodies.

OBJECTIVES: Antigen expression intensity is becoming important for decision-making in relation to monoclonal antibody therapy. By quantifying CD20, CD22 and CD52 expression on chronic lymphocytic leukemia and normal (control) B cells, over time. The effect of Interleukin-4 therapy on CD20 antigen intensity on B-CLL cells in vivo was also determined. METHODS: Lymphocytes were purified at weeks 0, 4 and 8 from five B-CLL patients, five healthy volunteers and seven B-CLL patients receiving IL-4 therapy. The number of antigen receptor sites was calculated in molecules of equivalent soluble fluorochrome using flow cytometry. RESULTS: The mean number of CD20 receptors at baseline was significantly lower on B-CLL cells compared to normal B cells (8160 vs 87 046; P<0.0001). Similar results were obtained for CD22 (8630 vs 27 647; P<0.01), but not for CD52 (371 303 vs 409 484; P = 0.54). When soluble fluorochrome values at weeks 4 and 8 were analysed as change in per cent from baseline (delta%), there was <10 delta% variability in CD20 expression on control B cells, but considerable variability (22.5-67.5 delta%) on B-CLL cells. Expression of CD22 in CLL and control B cells varied by <15 delta%. CD52 on CLL B cells showed slightly greater variability (+/-35 delta%) than that of CD22 (+/-15delta%), but less than that of CD20. IL-4 therapy did not consistently increase the CD20 expression on B-CLL cells in vivo. CONCLUSION: Our data confirm differences in intensity between different target antigens on B-CLL cells, and draws attention to the fact that a substantial variability may occur over time, which may influence clinical decision-making. Caution must be taken when interpreting in vitro results on cytokine-mediated receptor intensity up-regulation.

Effects of eccentric exercise on the immune system in men.

The effects of eccentric exercise on changes in numbers of circulating leukocytes, cell activation, cell adhesion, and cellular memory function were investigated in 12 men, aged 22-35 yr. The immunologic effects of postexercise epidermal treatment with monochromatic, infrared light were also evaluated. Blood was drawn before and 6, 24, and 48 h after exercise for phenotyping and analysis of creatine kinase activity. There was an increase in leukocyte, monocyte, and neutrophil number, no change in the number of basophils, eosinophils, B cells, and T cells, and a decrease in natural killer cell number postexercise. Some markers of lymphocyte and monocyte activation remained unchanged or decreased, whereas the expression of adhesion molecules 62L and 11b increased on monocytes. It is concluded that eccentric exercise induced decreased activation, and increased cell adhesion capacity, of monocytes. Altered trafficking of cells between lymphoid tissue and blood, selective apoptosis, or attachment/detachment from the endothelial wall can explain the observed phenotypic changes. Treatment with monochromatic, infrared light did not significantly affect any of the investigated variables. Correlations between immunologic and physiological parameters indicate a role of the immune system in adaptation to physical exercise.

A new antigen/antibody system--the polymerized albumin and antialbumin antibodies.

Two new antigenic determinants have been identified on serum albumin molecules originating from hepatic patients. The same antigenic determinants may be induced on serum albumins of different species by in vitro aging or glutaraldehyde treatment. In patients with liver cell dysfunction the new antigenic sites of the albumin molecules induce the synthesis of specific antipolymerized albumin antibodies (AAA) identified by precipitation and hemagglutination methods. The anti-polymerized albumin precipitins (AA-P) reveal with high specificity and sensitivity the presence and severity of liver cell dysfunction. In patients with active liver diseases (acute viral hepatitis and chronic aggressive hepatitis), both precipitins and agglutinins show high titers, while in patients with severe liver diseases and long evolution or in old-agers AA-P positivity is seldom associated with anti-polymerized albumin agglutinins.

High correlations of anti-CMV titers with lymphocyte activation status and CD57 antibody-binding capacity as estimated with three-color, quantitative flow cytometry in blood donors.

Blood samples of 58 blood donors (ages 19-64 years, 31 males and 27 females) were investigated with three-color, quantitative flow cytometry. IgG anti-cytomegalovirus (CMV) was estimated with ELISA. Nineteen men and 21 women were found CMV-seropositive. The seropositive individuals (CMV-pos) compared to the seronegative ones (CMV-neg) presented no significant changes in the absolute number of lymphocytes or in the main lymphocyte subpopulations identified by CD20, CD3, CD4, and CD8. Nevertheless, CMV-pos showed significantly higher numbers of lymphocytes with the following phenotypes: CD4+HLA-DR+, CD8+HLA-DR+, CD8+CD45RA+ CD45RO+, and CD20+CD5+. The CD3+CD57+ subset of T lymphocytes was also significantly higher in CMV-pos (P < 0.0001), whereas the CD57+CD16 & CD56+CD3- NKC subset presented similar values in both groups. Higher values for the antibody-binding capacity of lymphocytes CD38 (P < 0.05) and CD45 (P = 0.02) antigens and of CD57 antigen on CD3+ lymphocytes (P < 0.0005) were found in the CMV-pos group. These results show that CMV upregulates the number of CD57 molecules on CD3+ and not on CD3- lymphocytes and suggest that the CD3 molecule or other molecules specifically present in T cells can play a role in CMV latency. The study also showed that CMV plays a major role in the maintenance of a fraction of lymphocyte in the activated state.

HBs antigen and blood glucose concentration.

HBsAg presence was studied by counterelectrophoresis (CEP) in 76 patients with liver cirrhosis and in 431 patients with diabetes mellitus. A striking correlation was found between high blood glucose values and HBsAg absence. Thus, HBsAg-negative cirrhotics (53%) had significantly higher levels of glycemia than the HBsAg-positive patients of the same age group, i.e. 95.75 +/- 6.36 ng/100 ml compared to 78.30 +/- 10.2/100 ml. This absence of HBsAg was also observed in all diabetics but one. As the incidence of HBsAg (CEP) was found to be of 3.63% in 253,460 subjects from different areas of Romania and 6.84% in 14,690 subjects with various non-hepatic diseases included, the chance of finding the 0.2% HBsAg incidence observed in the diabetics would be less than 0.0002 and 0.0001, respectively. The serum HBsAg absence in cirrhotics with high glycemia and in diabetics strongly incriminates the constant high concentrations of blood glucose as the main factor responsible for this negativity. The effect may be direct on virus replication, or indirect, by metabolically-induced hepatic dysfunction interfering with HBsAg secretion or excretion. The presence of high concentrations of glucose in cell culture media might explain the repeated failure of hepatitis B virus serial passage in tissue or organ culture.

Receptors for polymerized albumin on liver cells.

By the use of rabbit polymerized albumin labelled with fluorescein isothiocyanate, or coated on sheep red blood cells, specific receptors on rabbit liver cells were demonstrated. The possible biological role of these receptors is discussed.

Flow cytometry of blood and bone marrow cells from patients with hairy cell leukemia: phenotype of hairy cells and lymphocyte subsets after treatment with 2-chlorodeoxyadenosine.

By flow cytometry and an extensive set of markers, we characterized leukemic cells from the blood and bone marrow of 68 symptomatic patients with hairy cell leukemia (HCL). Hairy cells identified in the large cell gate always expressed CD19, CD20, HLA-DR, CD45RA, and B-ly 7. Other markers were occasionally expressed, such as CD38, CD45RO, CD23, CD15, CD4, CD5, and CD10 (expressed on more than 20% of the hairy cells in 44%, 25%, 21%, 18%, 12%, 10%, and 5% of evaluated cases, respectively). During treatment with 2-chlorodeoxyadenosine (CdA), the median lymphocyte counts decreased from 2,000/microL to 300/microL. Flow cytometry was repeated at the nadir (n = 24) of lymphocyte counts, at 3 months (n = 46), at 6 months (n = 50), at 1 year (n = 39), and at 2 years (n = 12) after treatment. The initial decrease of CD8+ and CD20+ cells was greater than that of CD4+ and natural killer (NK) cells, leading to an increasing CD4/CD8 ratio. Median nadir values of CD4+, CD8+, CD20+, and NK cells were 128/microL, 78/microL, 10/microL, and 13/microL, respectively. The subsequent recovery was quicker for CD8+ and NK cells, leading to a normalization within 3 months, whereas CD20+ and CD4+ cells required 1 or 2 years to enter the normal range. The CD4/CD8 ratio thus decreased after the nadir and remained less than 1. CD45RA+ CD4 cells and CD45RA+/CD45RO+ double-positive cells were less affected by CdA. Activated T cells, ie, HLA-DR+ cells, rarely decreased below the normal range and often recovered with an overshoot. CD10+ cells increased in the bone marrow posttreatment as an indication of normal B-cell regeneration in 16 of 36 (44%) patients. The quick regeneration of certain lymphoid subsets might explain the lack of late infections in CdA-treated HCL patients.

Urinary excretion of albumin and beta 2-microglobulin in a population from an area where Balkan nephropathy is endemic.

The urinary excretion of albumin and beta 2-microglobulin in a population of 294 persons, living in an area where Balkan nephropathy is endemic, has been studied. In fifty-six (about 19%) of the subjects the beta 2-microglobulin concentration was above the +2 SD level for a reference group of healthy individuals from non-endemic areas. Albumin elevation was found in forty-four (about 15%) of the cases. In twenty-one of the subjects the urinary concentration of both beta 2-microglobulin and albumin were increased, in sixteen of these cases the relationship between the two proteins was consistent with tubular proteinuria. An increased beta 2-microglobulin excretion is considered to be a sign of Balkan nephropathy. Radioimmunoassay of the protein is sensitive enough to detect tubular proteinuria at an early stage and is suggested as a suitable screening test for the disease.

Deeper insight into HBsAg--anti-albumin antibody correlations.

The presence and titers of anti-albumin antibodies (AAA)--ie, precipitins (AA-IP) and agglutinins (AA-Aggl)--and the serum concentrations of immunoglobulins and albumin were determined in 210 asymptomatic carriers of HBsAg grouped according to HBsAg titer and subtype. A different immunologic pattern was observed in the HBsAg/ad and HBsAg/ay carriers, the results suggesting an increased aggressivity for the HBsAg/ay subtype which was evident in subjects with lower HBs antigenemia and characterized by higher concentrations of IgG and IgM and rises in AA-P titers. A positive linear correlation was found between HBsAg and AA-Aggl titers; the carriage of HBsAg/ad was associated with significant higher values of AA-Aggl than of HBsAg/ay. These subtle correlations between HBsAg titer and subtype and AAA suggest that the human serum albumin (HSA) known to exist on the HBsAg particles may be similar in a modified form, to that representing AAA specificity.

ABO blood groups and HBs AG subtypes and titers in healthy carriers.

The investigation covered 457 'healthy' HBsAg carriers detected following a screening by CEP. HBsAg in the sera of these subjects were subtyped by ID and titrated by CEP. The results obtained were analyzed according to the cases investigated. Differences were found between the distribution of blood groups among the normal unselected population and among 'healthy' carriers, where the incidence of AB subjects was much higher. The difference was more marked for the carriers of HBsAg/ad.