Rabbit Lymphocytes Research Articles

Establishment of anti-asialo-GM1 rabbit monoclonal antibodies capable of reducing natural killer cell activity in mice.

Rabbit anti-asialo-GM1 (ASGM1) serum or polyclonal antibodies can eliminate mouse splenic natural killer (NK) cell activity in vitro and in vivo. We developed rabbit monoclonal antibodies (mAbs) against ASGM1 using a single-cell analysis and isolation system. Five mAbs (GA109, GA115, GA116, GA131, and GA134) that were reactive to ASGM1 were isolated from the spleen lymphocytes of rabbits immunized with ASGM1. Enzyme-linked immunosorbent assay and thin-layer chromatography immunostaining results showed that the mAbs strongly reacted with ASGM1. Two mAbs (GA116 and GA134) reacted exclusively with ASGM1, whereas three mAbs (GA109, GA115, and GA131) showed slight or considerable cross-reactivity with GM1. The administration of the mAbs (4-20 μg) to BALB/c mice completely abolished NK cell activity in vivo. The anti-ASGM1 rabbit mAbs obtained in this study may provide a useful and reproducible tool for various future studies, such as depleting NK cell activity to enhance xenograft engraftment in mouse models.

Crosstalk between Apoptosis and Cytotoxic Lymphocytes (CTLs) in the Course of Lagovirus Europaeus GI.1a Infection in Rabbits.

Lagovirus europaeus is a single-stranded RNA virus causing an acute fatal disease in wild and domestic rabbits around the world. Studies have shown that the pivotal process impacting the immune response against the disease is apoptosis, registered mainly in hepatocytes and in peripheral blood, together with an increased number of cytotoxic lymphocytes (CTLs). It is known that cytotoxic lymphocytes can induce target cells to undergo apoptosis on the pseudoreceptor pathway, such apoptosis having been found in several acute and chronic viral infections. The study aimed to assess the crosstalk between the apoptosis of peripheral blood lymphocytes and CD8+ T lymphocytes (as CTLs) in rabbits infected with 6 Lagovirus europaeus GI.1a viruses. Sixty rabbits of Polish hybrid breed comprising both sexes and weighing 3.2-4.2 kg were the experimental group, and an identical group was the control. Each of the six GI.1a Lagovirus europaeus viruses was inoculated into ten experimental rabbits. Control rabbits received glycerol as a placebo. Flow cytometric analysis was performed on blood from the study and control group animals for peripheral blood lymphocyte apoptosis and CTL percentage determination. The activation of apoptosis in peripheral blood lymphocytes was recorded from 4 h post inoculation (p.i.) up to 36 h p.i. The percentage of CTLs in the total blood pool decreased from 8 to 36 h p.i. A negative correlation between apoptosis of lymphocytes and the number of CTLs was proven. This may be the first evidence of virus-induced CTL apoptosis in Lagovirus europaeus GI.1a infection.

Effect of allogeneic blood transfusion on neutrophil functional activity and lymphocyte cytotoxicity in recipient rabbits

The relevance of this paper is that transfusion of allogeneic blood to recipient animals is always associated with immunological risks. In this regard, the purpose of this study was to assess the state of phagocytic activity of blood neutrophils by indicators of phagocytic index, phagocytic number, and oxygen-dependent bactericidal activity, as well as to establish changes in antibody-dependent cytotoxic activity of lymphocytes in recipient rabbits during allogeneic whole blood transfusion. Modelling of blood transfusions was performed on five clinically healthy rabbits by intravenous administration of whole blood at the rate of 5.5 ml/kg of body weight. Blood samples were taken from animals on Days 3, 7, and 23 after blood transfusion. Neutrophil populations were obtained from blood samples by centrifugation on a double density gradient of 1.077 and 1.093 Ficoll-Verografin. The absorption activity of phagocytes was determined in a microscopic test. To investigate the oxygen-dependent bactericidal activity of neutrophils, a spontaneous test with nitroblue tetrazolium was performed. Antibody-dependent cytotoxic activity of lymphocytes was investigated by colorimetric method. It was found that after the transfusion of whole blood, the phagocytic activity of neutrophils increases with a simultaneous decrease in their absorption capacity. On Days 3 and 7, the results of the spontaneous test with nitroblue tetrazolium decreased. This indicates inactivation of the oxygen-dependent bactericidal activity of neutrophil granulocytes during the first phase of post-transfusion immunological reactions. On Day 23, there was an increase in the values of the indicators of the spontaneous test with nitroblue tetrazolium, which indicates the activation of the bactericidal properties of phagocytes. It was found that on Day 3, the antibody-dependent cytotoxic activity of lymphocytes significantly decreased relative to the initial state, and on Days 7 and 23, it increased. An increase in the antibody-dependent cytotoxic activity of lymphocytes should be associated with the active synthesis of antibodies of the late phase of the immune response. Consequently, transfusion of allogeneic blood causes an immune response in recipient rabbits, without causing immediate and long-term transfusion reactions (changes in heart rate, respiratory rate, body temperature). The obtained results are of practical value both for scientists and practising doctors who use transfusion of whole blood and its components to animals with acute anaemia, impaired functional activity of blood coagulation factors, parasitic, and oncological diseases

Changes in rabbit lymphocyte subpopulations and activation following long-term administration of meloxicam

Meloxicam is a commonly used analgesic in rabbits. However, its possible impact on lymphocyte subpopulations remained unknown. The aim of the study was to investigate a possible effect of long-term administration of meloxicam on rabbit lymphocyte subpopulations. The study included 8 rabbits given meloxicam orally once daily (1 mg/kg BW) for 14 days and 8 rabbits as a control group. Peripheral blood samples were collected on day 0 (before the first dose of meloxicam), day 3, 7 and 14. Samples were evaluated with a haematology analyser and a flow cytometer. A significant decrease in T: B cell ratio was found in all samples taken during meloxicam administration compared to day 0, as well as in comparison with the control group (P < 0.01). A significant increase (P < 0.05) in proportion of CD5 +CD8 + lymphocytes occurred by day 3. Subsequently, although the values slightly decreased, they still remained elevated throughout all the experiment compared to the values from day 0 (P < 0.05). A slight decrease in T and B cell activation (CD5 +CD25 + and IgM+CD25 +) noticed by day 3, declined during the next days of administration and became more and more significant (finally, P = 0.0078). Since a high significant decrease (P < 0.01) in both T and B cell activation as well as a significant increase (P < 0.05) in CD5 +CD8 + T cells proportion were observed after meloxicam administration, a predicted effect of long-term administration of meloxicam on rabbit lymphocytes was confirmed.

SSEA-4 Antigen Is Expressed on Rabbit Lymphocyte Subsets

SSEA-4 antigen can be mainly found in embryos and embryonic stem cells. However, its expression has been observed also in adult stem and progenitor cells, or even in some differentiated cells. Moreover, we found a considerable number of SSEA-4 positive (SSEA-4+) cells within the rabbit peripheral blood and bone marrow mononuclear cells (PBMCs and BMMCs) in our previous study. Since no information about such cells can be found anywhere in the literature, the aim of this study was to identify their origin. At first, phenotypic analyses of fresh rabbit PBMCs and BMMCs were performed using flow cytometry and specific antibodies against SSEA-4 and leukocyte subsets. Then, SSEA-4+ were enriched using magnetic activated cell sorting (MACS) and analyzed for their phenotype using qPCR. We found significant SSEA-4+ cell population in PBMCs (~50%) and BMMCs (~20%). All those cells co-expressed CD45 and a majority of them also expressed B-cell marker (IgM; 50% of SSEA-4+ PBMCs and 60% of SSEA-4+ BMMCs). Increased (p &lt; 0.05) expression of SSEA-4, CD45 and B-cell markers (IgM, CD79α and MHCII) were also noticed by qPCR in SSEA-4+ cells enriched via MACS (with efficiency over 80%). Both methods did not detect significant expression of monocyte or T-cell markers. In conclusion, SSEA-4+ cells in rabbit blood and bone marrow are of hematopoietic origin and probably belong to B-lineage cells as possessing the phenotype of B lymphocytes. However, the true function of SSEA-4 antigen in these cells should be explored by further studies.

Development of a time-resolved fluoroimmunoassay for measuring plasma growth hormone in Nile tilapia (Oreochromis niloticus)

Growth hormone is a hormone secreted from the pituitary and is involved in the regulation of most major physiological processes such as growth, development and metabolism. Therefore, an accurate and sensitive detection method is needed for the detection of tilapia serum Gh level. Phage display technology is widely used in the expression of antibody fragments, in which fragments of antibodies are expressed as a fusion with phage proteins and are displayed on the phage surface for easy screening. Time-resolved fluorescence immunoassay (TRFIA) is a microanalysis method developed nearly two decades ago and is one of the most sensitive analytical techniques. With the use of a special lanthanide, the detection background can be distinguished, which can greatly improve the sensitivity of detection.In this report, we cloned the VH and VL DNA fragments from the lymphocytes of rabbits immunized with recombinant Gh and assembled them with a linker to form a single-chain variable fragment (scFv) gene pool. Using phage display technology, we isolated scFv DNA fragments from the pool, which encode a protein that specifically binds to tilapia Gh. We then established Eu-DTTA-based TRFIA for measuring plasma Gh in tilapia. The sensitivity of double antibody sandwich Gh-TRFIA was 0.225 ng/ml, and the linear range of the standard curve was 0.225–250 ng/ml. The intra- and interassay coefficients of variation (CVs) were <9.1 and <4.5%, respectively. The cross-reactivities (CRs) of 1 μg/ml recombinant tilapia somatolactin (rtSl), prolactin (rtPrl) and thyroid-stimulating hormone beta subunit (rtTshb) were 0.042%, 0.472% and 0.036%, respectively. The sensitivity of direct competitive Gh-TRFIA was 0.208 ng/ml, and the linear range of the standard curve was 0.208–500 ng/ml. The intra- and interassay CVs were <4.8 and <7.1%, respectively. The CRs of 1 μg/ml rtSl, rtPrl and rtTshb were 0.041%, 0.079% and 0.073%, respectively.In conclusion, Gh-TRFIA is a safe (no concerns about radioactive isotopes), economical, and efficient detection method for the quantification of plasma Gh. Thus, the application of phage display technology for antibody screening and the use of TRFIA for tilapia Gh detection are conducive to research in the field of fish endocrinology.

Increased expression of blood muscarinic receptors in patients with reflex syncope.

AimsPathophysiology of reflex syncope is not fully understood but a vagal overactivity might be involved in this syncope. Previously, overexpression of muscarinic M2 receptors and acetylcholinesterase was found in particular in the heart and in lymphocytes of rabbits with vagal overactivity as well as in hearts of Sudden Infant Death Syndromes. The aim of this present study was to look at M2 receptor expression in blood of patients with reflex syncope. The second objective was to measure acetylcholinesterase expression in these patients.Methods and results136 subjects were enrolled. This monocenter study pooled 45 adults exhibiting recurrent reflex syncope compared with 32 healthy adult volunteers (18–50 years) and 38 children exhibiting reflex syncope requiring hospitalization compared with 21 controls (1–17 years). One blood sample was taken from each subject and blood mRNA expression of M2 receptors was assessed by qRT-PCR. Taking into account the non-symmetric distributions of values in both groups, statistical interferences were assessed using bayesian techniques. A M2 receptor overexpression was observed in adult and pediatric patients compared to controls. The medians [q1;q3] were 0.9 [0.3;1.9] in patients versus 0.2 [0.1;1.0] in controls; the probability that M2 receptor expression was higher in patients than in controls (Pr[patients>controls]) was estimated at 0.99. Acetylcholinesterase expression was also increased 0.7 [0.4;1.6] in patients versus 0.4 [0.2;1.1] in controls; the probability that acetylcholinesterase expression was higher in patients than in controls (Pr[patients>controls]) was estimated at 0.97. Both in adults and children, the expression ratio of M2 receptors over acetylcholinesterase was greater in the patient group compared with the control group.ConclusionM2 receptor overexpression has been detected in the blood of both, adults and children, exhibiting reflex syncope. As in our experimental model, i.e. rabbits with vagal overactivity, acetylcholinesterase overexpression was associated with M2 receptor overexpression. For the first time, biological abnormalities are identified in vagal syncope in which only clinical signs are, so far, taken into account for differential diagnosis and therapeutic management. Further work will be needed to validate potential biomarkers of risk or severity associated with the cholinergic system.

Antagonism of Dexamethasone to Radiation Bystander Effect and Its Re-transmission

Background: In clinical radiotherapy, in addition to the symptoms of radiation damage in the irradiated area, normal tissues can produce damage other than direct irradiation due to bystander effects. We often use dexamethasone to relieve symptoms, thereby protecting the surrounding normal tissues, and may also protect the tissues outside the target area. Objectives: To observe the antagonism of dexamethasone on radiation bystander effect and its Re-transmission. Materials and methods: After 6MV X-ray irradiation of rabbit, the plasma, the first generation conditioned medium, was prepared to treat rabbit lymphocyte, cultured for 24 hours, 24 hours later, lymphocytes were collected, and the second generation conditioned medium was prepared with the lymphocyte secretion to treat new rabbit lymphocyte, and the apoptotic rate of effecter lymphocyte was detected. Dexamethasone was used as a parallel treatment control in all experimental groups. Results: The first and second generations of conditioned medium can stimulate lymphocyte apoptosis. Dexamethasone could reduce the apoptotic level of rabbit lymphocytes treated with the first and second generation bystander conditioned medium from 21.087% to 14.957%, 28.695% to 20.205%, respectively. Conclusion: Under certain conditions, dexamethasone can antagonize the injury of lymphocyte by radiation bystander effect, and can also antagonize the re-transmission of radiation bystander effect.

Macaca arctoides gammaherpesvirus 1 (strain herpesvirus Macaca arctoides): virus sequence, phylogeny and characterisation of virus-transformed macaque and rabbit cell lines.

Herpesvirus Macaca arctoides (HVMA) has the propensity to transform macaque lymphocytes to lymphoblastoid cells (MAL-1). Inoculation of rabbits with cell-free virus-containing supernatant resulted in the development of malignant lymphomas and allowed isolation of immortalised HVMA-transformed rabbit lymphocytes (HTRL). In this study, the HVMA genome sequence (approx. 167 kbp), its organisation, and novel aspects of virus latency are presented. Ninety-one open reading frames were identified, of which 86 were non-repetitive. HVMA was identified as a Lymphocryptovirus closely related to Epstein-Barr virus, suggesting the designation as 'Macaca arctoides gammaherpesvirus 1' (MarcGHV-1). In situ lysis gel and Southern blot hybridisation experiments revealed that the MAL-1 cell line contains episomal and linear DNA, whereas episomal DNA is predominantly present in HTRL. Integration of viral DNA into macaque and rabbit host cell genomes was demonstrated by fluorescence in situ hybridisation on chromosomal preparations. Analysis of next-generation sequencing data confirmed this finding. Approximately 400 read pairs represent the overlap between macaque and MarcGHV-1 DNA. Both, MAL-1 cells and HTRL show characteristics of a polyclonal tumour with B- and T-lymphocyte markers. Based on analysis of viral gene expression and immunohistochemistry, the persistence of MarcGHV-1 in MAL-1 cells resemble the latency type III, whereas the expression pattern observed in HTRL was more comparable with latency type II. There was no evidence of the presence of STLV-1 proviral DNA in MAL-1 and HTRL. Due to the similarity to EBV-mediated cell transformation, MarcGHV-1 expands the available in vitro models by simian and rabbit cell lines.

Effect of graded levels of ginger rhizome (Zingiber officinale) meal on haematology of pubertal New Zealand white rabbits

Forty-eight sexually matured (7 to 8 months old) healthy New Zealand White rabbits consisting of male and female of equal sexes were used to determine the effect of graded levels of ginger rhizome meal on haematological parameters. The rabbits were divided into 4 treatment groups, identified as T1, T2, T3 and T4 and randomly assigned to four treatment diets in a randomized complete block design (RCBD). Each of the treatment groups consisted of 12 rabbits (6 does and 6 bucks) replicated 3 times with 4 rabbits per replicate. The levels of inclusion of the ginger rhizome meal were 0.00, 5.00, 10.00 and 15.00% identified as T1, T2, T3 and T4 respectively. Treatment one (T1) which contained no ginger rhizome meal served as the control. The rabbits were fed twice daily, in the morning and evening. Water was given ad libitum to the rabbits. After 30 days of the feeding trail, blood samples were randomly collected from 12 rabbits (6 does and 6 bucks) in each treatment group for hematological evaluation. The results showed that there were significant differences (P 0.05) among the treatment groups in red blood cells (RBC), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC), basophils and monocyte values. The results further showed that the HB, PCV, RBC, WBC, neutrophil and lymphocyte values of the rabbits were significantly (P<0.05) influenced by sexes. Haemoglobin (12.72g/dl) PCV 37.87% RBC 5.54 (x1012/L) and lymphocytes (47.67%) of the male rabbits (bucks) were significantly (P<0.05) higher than their female (doe) counterparts 11.97 g/dl, 34.60%, 4.98 (x1012/L) and 39.00% respectively. Mean corpuscular volume (70.43 fl), WBC (9.07x109/L) and neutrophils (60.00%) of the female rabbits differed significantly (P<0.05) from their male counterparts 67.65 fl, 6.40x109/l and 51.33% respectively. The results of this study indicate that inclusion of ginger rhizome meal in the diets of the pubertal rabbits up to 5% improved the haematological indices of these rabbits.

Myxoma Virus M083 Is a Virulence Factor Which Mediates Systemic Dissemination.

Poxviruses are large, DNA viruses whose protein capsid is surrounded by one or more lipid envelopes. Embedded into these lipid envelopes are three conserved viral proteins which are thought to mediate binding of virions to target cells. While the function of these proteins has been studied in vitro, their specific roles during the pathogenesis of poxviral disease remain largely unclear. Here we present data demonstrating that the putative chondroitin binding protein M083 from the leporipoxvirus myxoma virus is a significant virulence factor during infection of susceptible Oryctolagus rabbits. Removal of M083 results in a reduced capacity of virus to spread beyond the regional lymph nodes and completely eliminates infection-mediated mortality. In vitro, removal of M083 results in only minor intracellular replication defects but causes a significant reduction in the ability of myxoma virus to spread from infected epithelial cells onto primary lymphocytes. We hypothesize that the physiological role of M083 is therefore to mediate the spread of myxoma virus onto rabbit lymphocytes, allowing these cells to disseminate virus throughout infected rabbits.IMPORTANCE Poxviruses represent both a class of human pathogens and potential therapeutic agents for the treatment of human malignancy. Understanding the basic biology of these agents is therefore significant to human health in a variety of ways. While the mechanisms mediating poxviral binding have been well studied in vitro, how these mechanisms impact poxviral pathogenesis in vivo remains unclear. The current study advances our understanding of how poxviral binding impacts viral pathogenesis by demonstrating that the putative chondroitin binding protein M083 plays a critical role during the pathogenesis of myxoma virus in susceptible Oryctolagus rabbits by impacting viral dissemination through changes in the transfer of virions onto primary splenocytes.

Recombinant Lactobacillus casei expressing Clostridium perfringens toxoids α, β2, ε and β1 gives protection against Clostridium perfringens in rabbits

The present study used Lactobacillus casei ATCC 393 as antigen delivery system to express C. perfringens toxoids α-β2-ε-β1 to construct the recombination Lactobacillus casei pPG-2-α-β2-ε-β1/L. casei 393. After being induced by 1% xylose, the specificity and integrity of recombinant strain were determined by Western-blotting. Rabbits as native animal model were immunized orally with pPG-2-α-β2-ε-β1/L. casei 393 and the titers of specific IgG and sIgA were determined by ELISA. The result showed that oral administration with the recombinants could elicit both local mucosal and systemic immune responses. The proliferation of spleen lymphocytes in rabbits immunized with pPG-2-α-β2-ε-β1/L. casei 393 was observed. Levels of IL-4 and IFN-γ produced were significantly higher in lymphocytes isolated from the vaccine group than those from the control groups. Flow cytometry assay showed that both the percentages of CD4+T cells and CD8+T cells from the vaccine group were significantly increased than the control groups. All these results showed that immunizing with recombinants can elicit both humoral immunity and cellular immunity. Besides, in order to determine the effectiveness of oral immunization with pPG-2-α-β2-ε-β1/L. casei 393, rabbits of vaccine group and control groups were challenged with 1×LD100 unit of culture filtrate of C. perfringens type C and type D toxins respectively. After challenge, 100% of the immunized rabbits survived, while the rabbits of the control group were killed within 48h. Observation on histopathology showed that histopathological changes were obviously found in heart, liver, spleen, lung, kidney, intestine and brain of rabbits from the control groups, while no apparent histopathological change was observed in the vaccine group. All the results show that pPG-2-α-β2-ε-β1/L. casei 393 can eliciteffective immunoprotection against C. perfringens. All of these suggest that the use of pPG-2-α-β2-ε-β1/L. casei 393 can be regarded as candidate for the development of a vaccine against C. perfringens.

Effects of Oregano (Origanum vulgare L.) and Rosemary (Rosmarinus officinalis L.) Aqueous Extracts On in vitro Rabbit Immune Responses

In order to investigate the effects of some dietary phytoderivates (Origanum vulgare L. and Rosmarinus officinalis L.) on rabbit immune system, 100 New Zealand mixed-sex rabbits weaned at 30 days of age were split into homogeneous groups submitted to the following dietary treatments: 1) Standard diet (C); 2) C +150 ppm Vit E (E); 3) C +0.2% oregano (O); 4) C +0.2% rosemary (R) and 5) C +0.1% oregano + 0.1% rosemary (OR). Blood samples were drawn from rabbits at 30 and 90 days of age (ten rabbits/diet group at Time 0 and 1, respectively) to investigate the blood lymphocyte subset evolution, the in vitrolymphocyte proliferation (in presence or absence of mitogens) responses and the Immunoglobulin M(IgM) concentration in the supernatants of lymphocyte cultures. A diet effect was observed in the O and OR groups, where the lymphocytes proliferation responses to pokeweed mitogen (PWM; ***P<0.001, in the O group) or the interleukin-2 production (IL-2; ***P<0.001, for both groups) at Time 1 were significantly higher. The IgM levels were systematically higher in the O, OR and E cells culture supernatants (**P<0.01). Age did not affect the rabbit lymphocyte subset evolution nor the in vitrolymphocyte proliferation. Data obtained in the present study show that rabbit’s dietary supplementation with oregano elicits positive effects on the adaptive immune response.

English

The aim of this study was to investigate the immunomodulatory activities of protein fractions isolated from Delphinium staphysagria seeds used in Moroccan traditional medicine. D. staphysagria protein extract has been separated on sephadex column chromatography. The immunomodulatory activities were tested using primary culture of rabbit lymphocytes (splenocytes and thymocytes), by evaluation of macrophage phagocytosis and by studying the hemagglutination effect of proteins separated by chromatography. Three protein fractions were isolated on sephadex column. At 0.1mg/ml, these fractions didn’t show any cytotoxic effect against lymphocytes. The fraction F1 with the high molecular weight of 115 kDa exhibited a mitogenic action on splenocytes and positive hemagglutinating activity associated to inhibition of macrophage phagocytosis capacity. The second protein fraction F2 was about 11 kDa inhibited phagocytosis without affecting lymphocytes proliferation. The third fraction F3 was the smallest with 2.25 kDa indicated a stimulatory effect on both splenocytes and thymocytes proliferation without hemagglutinating activity but inducing decrease of macrophage phagocytosis. In conclusion, protein fractions isolated from D. staphysagria have no cytotoxic effect. The fraction F3 acted as an immunostimulating by enhancing proliferation of thymocytes and splenocytes while the F1 stimulated preferentially splenocytes via probably lectin pathway. All fractions studied decreased phagocytosis impairing the antigen presentation to lymphocytes. Key words: Delphinium staphysagria, protein fractions, immunomodulation, mitogenic effect, hemagglutination, MTT assay.

Hematological and serum biochemistry comparison between cotton hair thread and silk suture on surgical skin wounds of rabbits

A total of 12 rabbits randomly assigned into three groups (I, II, and III) of 4 rabbits each were used. A 6-cm-long paralumber skin incision was aseptically performed in groups I and II using xylazine (0.5 mg/kg) and ketamine (22 mg/kg) as premedicant and anesthetic, respectively. The incised skins in group I rabbits were apposed with conventional silk suture, while the incised skins in the group II rabbits were approximated with cotton hair thread (CHT). Group III served as a control (no surgery). The hematological and serum biochemistry responses following the use of these sutures were evaluated. Packed cell volume (PCV), red blood cell (RBC) count, total and differential white blood cell (WBC) counts, total protein, and albumin and globulin levels were determined using standard methods at presurgery (day 0) and on postoperative days 1, 3, 7, 10, 14, and 21 in all the groups. Hematological and serum biochemistry parameters were analyzed using one-way analysis of variance (ANOVA). The means of PCV and RBCs in rabbits in groups I, II, and III showed no significant variations (p > 0.05) throughout the study. The WBC count increased significantly (p 0.05) throughout the study. However, at day 3 postsurgery, the means of lymphocytes in rabbits in groups I and II increased significantly (p 0.5), but they vary and are significantly higher (p 0.05) throughout the study. Cotton hair Thread elicited insignificant hematological and serum biochemical changes compared to the conventional silk suture material.

The cytogenetic action of ifosfamide, mesna, and their combination on peripheral rabbit lymphocytes: an in vivo/in vitro cytogenetic study

Ifosfamide (IFO) is an alkylating nitrogen mustard, administrated as an antineoplasmic agent. It is characterized by its intense urotoxic action, leading to hemorrhagic cystitis. This side effect of IFO raises the requirement for the co-administration with sodium 2-sulfanylethanesulfonate (Mesna) aiming to avoid or minimize this effect. IFO and Mesna were administrated separately on rabbit's lymphocytes in vivo, which were later developed in vitro. Cytogenetic markers for sister chromatid exchanges (SCEs), proliferation rate index (PRI) and Mitotic Index were recorded. Mesna's action, in conjunction with IFO reduces the frequency of SCEs, in comparison with the SCEs recordings obtained when IFO is administered alone. In addition to this, when high concentrations of Mesna were administered alone significant reductions of the PRI were noted, than with IFO acting at the same concentration on the lymphocytes. Mesna significantly reduces IFO's genotoxicity, while when administered in high concentrations it acts in an inhibitory fashion on the cytostatic action of the drug.

Seasonal influence on mitogen and cyclosporin responses of peripheral blood lymphocytes

The immune response and lymphocyte activation in particular are affected by environmental factors. In vivo and in vitro experiments demonstrate variability in lymphocyte activation according to seasonal changes. This study focused on the effects of season on the ex vivo mitogen-induced activation of lymphocytes from peripheral blood of healthy humans living in a temperate climate, as well as the ex vivo lymphocyte activation of rabbits living under constant laboratory conditions. The possible impact of season on the action of the immunosuppressant drug cyclosporin A (CsA) on lymphocyte activation was investigated in both species. Cultured peripheral blood lymphocytes from human donors (n=13, 22-63years of age) and from animals housed under 12:12hour light:dark cycle were stimulated with phytohemagglutinin (PHA) in the absence or presence of 10 and 25μg/mL CsA. Lymphocyte activation was assessed by morphometric analysis under a light microscope. Percentages of unactivated lymphocytes, activated lymphoblasts and aberrant cells reflecting cytotoxicity were determined. Human lymphocytes demonstrated a significant decrease in response to PHA during the winter months, in comparison to the rest of the year. In contrast, the peripheral blood lymphocytes of rabbits housed under constant conditions did not demonstrate similar variations in response to PHA stimulation. The immunosuppressive action of cyclosporin A on this experimental model was unaffected by the observed seasonal variation in mitogen response in humans. These findings may guide research towards the identification of factors associated with the seasonality of the immune response and its potential influence on therapeutic interventions.

Apoptosis of peripheral blood leucocytes in rabbits infected with different strains of rabbit haemorrhagic disease virus.

The pathogenicity of RHDV (rabbit haemorrhagic disease virus) is mainly associated with its affinity to blood vessels, with causing disseminated intravascular coagulations (DIC), and with the stimulation of the host immune system. Moreover, there are implications suggesting that apoptosis may be a pivotal process in understanding the basis of viral haemorrhagic disease in rabbits - a serious infectious disease causing mortality to wild and domestic rabbits. The aim of this study is to evaluate, by means of flow cytometry, the dynamics of apoptosis in peripheral blood granulocytes and lymphocytes in rabbits experimentally infected with seven different strains of RHDV and so-called antigenic variants of RHDV denominated as RHDVa, i.e.: Hungarian 24V/89, 1447V/96, 72V/2003; Austrian 01-04, 237/04, V-412 and French 05-01. The results showed that all of the RHDV and RHDVa strains cause an increase in the number of apoptotic cells throughout the infection, which might indicate the need for further analysis of the importance of this process.

A Novel Rabbit Immunospot Array Assay on a Chip Allows for the Rapid Generation of Rabbit Monoclonal Antibodies with High Affinity

Antigen-specific rabbit monoclonal antibodies (RaMoAbs) are useful due to their high specificity and high affinity, and the establishment of a comprehensive and rapid RaMoAb generation system has been highly anticipated. Here, we present a novel system using immunospot array assay on a chip (ISAAC) technology in which we detect and retrieve antigen-specific antibody-secreting cells from the peripheral blood lymphocytes of antigen-immunized rabbits and produce antigen-specific RaMoAbs with 10–12 M affinity within a time period of only 7 days. We have used this system to efficiently generate RaMoAbs that are specific to a phosphorylated signal-transducing molecule. Our system provides a new method for the comprehensive and rapid production of RaMoAbs, which may contribute to laboratory research and clinical applications.

Apoptosis of peripheral blood leukocytes from rabbits infected with non-haemagglutinating strains of rabbit haemorrhagic disease virus (RHDV)

The report demonstrates that the induction of apoptosis in peripheral blood granulocytes and lymphocytes of rabbits infected with three non-haemagglutinating RHDV strains (English Rainham, German Frankfurt, and Spanish Asturias) is a crucial determinant of the pathogenesis of rabbit haemorrhagic disease. Apoptosis was measured by flow cytometric detection of caspase activity. These studies demonstrated that the investigated RHDV (rabbit haemorrhagic disease virus) viral strains affected leukocyte apoptosis to varying degrees. Enhanced leukocyte apoptosis was detected between 4 and 36h after infection and was more pronounced in lymphocytes than in granulocytes. The data presented here thus provide a preliminary understanding of the kinetics of apoptosis in leukocytes of rabbits infected with RHDV.