An influence of immunomodulation on Th1 and Th2 immune response in endometriosis in an animal model.

AIM: To assess the role of the Th1 and Th2 cellular response in the etiology of endometriosis observed in a rat model, with the use of the RESAN immunomodulator. MATERIALS AND METHODS: A comparative analysis of cytokines in blood serum typical of Th1 (TNF-α and INF-γ ) and Th2 (IL-4, IL-6, IL-10) cell response in groups of rats, in which RESAN preparation was used as prophylaxis (Gr. I) or treatment (Gr. II) of endometriosis. RESULTS: The results indicated an increase in the level of cytokines in blood serum typical of Th2 cell response by comparing the second and third stages of the experiment in the second group of rats and a decrease in IL-4 and IL-10 between III and IV stages. There was a significant difference in cytokine levels during the third stage of the experiment by comparing I and II groups of rats. In the III group of rats, levels of IL-10 significantly increased between the II and III stages of the experiment. CONCLUSION: RESAN preparation shows Th2 cell response, inhibiting the development of endometriosis in a rat model. Due to successful prophylactic action, one may speculate that RESAN vaccine may be effective as a complementary treatment after surgical excision.

Skin cancer following kidney transplantation: a single-center experience.

One of the major problems associated with prolonged immunosuppression is a high occurrence of skin malignancies among kidney recipients. Studies have shown that nonmelanoma skin cancer is the most frequently occurring tumor after organ transplantation. The aim of this study was to determine the incidence of and identify possible risk factors for skin malignancies among a population of kidney recipients. This retrospective, single-center cohort comprised 1672 patients transplanted from 1994 to 2011. Only patients with a confirmed diagnosis of skin cancer were selected for medical records review. Among 836 kidney transplant recipients remaining under our care since 1994, skin malignancies were diagnosed in 16 patients (1.9%). The histological diagnoses included squamous cell carcinoma (n=8; 50.0%); basal cell carcinoma (n=6; 37.5%) or malignant melanoma (n=2; 12.5%). The slightly lower incidence of skin malignancies noted in our study compared with other reports might result from differences in the length of follow-up. Some patients diagnosed with skin cancer were treated in local dermatology clinics. Also, a lower exposure to the sun characteristic for the latitude and differences in immunosuppressive therapies could be partially responsible for the lower skin cancer incidence. We also did not observe any association between other reported risk factors, such as age, human leukocyte antigen mismatch, duration of pretransplant hemodialysis, particular immunosuppressive therapies and the skin cancer occurrence among our kidney recipients.

Possible defect of T suppressor cell subpopulation in patients with kidney acute rejection.

CD8(+)CD28(-) forkhead box P3 (Foxp3(+)) T suppressor (Ts) lymphocytes are antigen-specific cells capable of inducing tolerogenic antigen-presenting cells by up-regulation of inhibitory receptors immunoglobulin-like-transcripts -3 and -4 and down-regulation of costimulatory molecules. Our study sought to investigate the relation between the level of peripheral CD8(+)CD28(-)Foxp3(+) Ts cells and kidney allograft outcomes. The project included 44 kidney transplantation patients. During the 6-month period following transplantation an acute rejection episode (ARE) was diagnosed in 11 patients based on biopsy results using the Banff criteria. Peripheral blood samples collected at 1 day before as well as 14 and 30 days after transplantation were tested for CD8(+)CD28(-)Foxp3(+) T cells by means of flow cytometry. Values were considered significant when P < .05. Cytometric analysis did not show significant differences between the groups in pretransplant levels of peripheral CD8(+)CD28(-)Foxp3(+) Ts cells (P > .05); however, the posttransplantation analysis showed a higher mean level of Ts cells in nonrejection (NONARE) versus acute rejection (ARE) patients (P < .0001). This observation suggested that dysfunction of CD8(+)CD28(-)Foxp3(+) Ts cells observed in ARE patients may contribute to these episodes. Interestingly, we observed similar results with respect to peripheral CD4(+)CD25(+)Foxp3(+) T regulatory cells in ARE patients, suggesting impairment of immunoregulatory mechanisms (especially within the inducible Foxp3 system) in this group, leading to acute renal allograft rejection episodes.

Distinct cytokine patterns in different states of kidney allograft function.

Cytokines are crucial inflammatory mediators involved in the development of immune response leading to allograft rejection. We investigated the cytokine patterns in patients sera from cases of acute rejection episodes (ARE), chronic rejection (CR), and long-term stable courses (STABLE). The project included 20 patients with ARE, 20 with CR, and 15 with at least a 5-year stable course. Serum samples collected at the time of rejection diagnosis were cytometrically tested for concentrations of interleukin (IL) 2, IL-4, IL-6, IL-10, interferon (IFN) gamma, and tumor necrosis factor alpha. No significant differences between investigated groups were observed before transplantation (P > .05). Significant differences were observed among the groups in serum levels of IFN-gamma, IL-4, IL-6, and IL-10. Our data suggested that distinct serum cytokine patterns were present among various states of kidney allograft function. ARE was characterized by a mixed cytokine pattern with elevated IL-10 and IFN-gamma compared with the STABLE patients. The cytokine pattern in CR patients, in turn, was characterized by elevated levels of IL-4, IL-6, and IL-10 and decreased levels of IFN- gamma compared with both STABLE and ARE subjects. Our results suggested that the T(H)2 response may contribute to the initiation and/or maintenance of CR, because IL-4, IL-6, and IL-10 serve as growth and differentiation factors for B cells to increase antibody production. We also observed up-regulated production of IFN-gamma and down-regulation of T(H)2 cytokines among patients with stable long- term graft function.

The role of Foxp3+ regulatory T cells in kidney transplantation.

Our project aimed to investigate the relation between the level of pretransplantation and posttransplantation peripheral CD4(+)CD25(+)Foxp3(+) T-regulatory lymphocytes (Tregs) and the development of acute rejection (AR) episodes in 44 patients after kidney transplantation. During the 6-month period following transplantation, AR was diagnosed in 11 patients. Peripheral blood samples were collected 1 day before and 10 days after transplantation and tested for concentrations of CD4(+)CD25(+)Foxp3(+) cells by means of flow cytometry. The pretransplantation analysis showed significantly lower mean levels of peripheral Tregs in AR patients versus control group (P < .05). A lower level of Tregs was also observed in nonrejection (NONAR) patients versus control group (P < .05); however, it was still higher than in the AR group (P < .05). The 10-day posttransplantation analysis showed a similar pattern; however, a significant increase in the concentration of peripheral Tregs in NONAR patients was observed (P < .05), whereas no change was recorded in AR patients (P > .05). We found lower pretransplantation levels of peripheral Tregs in both AR and NONAR groups, versus control group. The deficiency of peripheral Tregs in patients with end-stage renal failure might be due to the long-term inflammatory processes adversely affecting the peripheral regulatory mechanisms. However, significantly lower levels of Tregs observed in AR patients might also be related to genetic predispositions. Our observation suggests that the size and possibly the functionality of Tregs in the AR group was not sufficient to successfully control the immune response after kidney transplantation, leading to acute rejection episodes.

Role of TH1/TH2 cytokines in kidney allograft rejection.

One of the major issues in contemporary kidney transplantation is prevention of acute allograft rejection episodes (AREs). Cytokines are crucial mediators of immune reactions leading to AREs. We correlated serum Th1/Th2 cytokine concentrations with AREs. The project included 44 patients undergoing kidney transplantation. During the 3-month period following the transplantation, ARE was diagnosed in 11 patients. Serum samples collected 1 day before and 2, 7, 14, and 30 days after transplantation were tested for interleukin (IL)-2, IL-4, IL-5, IL-10, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha concentrations using flow cytometry. Nonrejection (NONAR) and rejection (ARE) groups of patients did not show significant differences in baseline demographic characteristics. We observed that higher pretransplantation serum levels of IFN-gamma (P = .000003) and IL-10 (P = .000001) were associated with AREs. Our analysis also showed slightly higher IL-4 serum levels among NONAR patients up to 7 days posttransplantation, followed by a drop in concentrations in NONAR patients. In contrast, there was a continuous increase among ARE patients. No significant differences were observed in plasma levels of IL-2, IL-5, IL-10, or TNF-alpha between the two groups. Higher pretransplantation levels of IFN-gamma and IL-10 observed in ARE patients indicated ongoing nondetected, probably nonspecific, inflammatory processes able to intensify an immune response directed against the transplanted organ leading to its acute rejection. Higher levels of IL-4 prior to and shortly after transplantation may have protective effects on graft survival. However, a prolonged, increased production of IL-4 after transplantation can also contribute to AREs.

No evidence of HTLV-I infection in patients with mycosis fungoides and Sezary syndrome.

The involvement of human T-cell lymphotropic virus type I (HTLV-I) in the etiology of cutaneous T-cell lymphomas (CTCL) is still controversial. The aim of the study was to evaluate the role of HTLV-I in the pathogenesis of mycosis fungoides (MF) and Sezary syndrome (SS) in Polish patients. The studied group consisted of 42 patients with MF, 5 with SS and 25 with chronic dermatitis. DNA was extracted from snap-frozen and paraffin-embedded skin biopsies and from peripheral blood. Polymerase chain reaction (PCR or nested PCR) was carried out for amplification of different regions of HTLV-I genome. Primer sets flanking pX, p 19, U5, tax and pol genes were used in the investigation. The presence of HTLV-I antibody was examined in 46 sera samples with the use of anti-HTLV-I/II EIA test. HTLV-I antibodies were not detected in any collected sera samples. PCR with two primer sets homologous to the pX region of HTLV-I showed negative results in all samples investigated. To confirm these results two other primer pairs specific for U5 and gag regions were designed. With these primer pairs no PCR product, except that in positive control, was observed. For more sensitive amplification a nested-PCR with pol and tax specific primers was performed. HTLV-I probably does not play an important role in the pathogenesis of MF in Polish patients.

The interactions of phthalocyanines with stimulated and resting human peripheral blood mononuclear cells.

The interactions of two metal-free phthalocyanines [(H2Pc) and Solar Pc (with four peripherical groups: SO2N(CH2CH2OH)2)] and of one metal substituted dye (CoPc) with resting and stimulated human peripheral blood mononuclear cells (PBMC) were compared. The absorption, fluorescence, photoacoustic and EPR spectra of both resting cells and cells stimulated by phytohaemagglutinin, incubated in dimethyl sulfoxide (DMSO) with very low or 95% water content and with or without dye addition, were measured. The fate of the light absorbed by the samples was investigated. It is known that singlet oxygen production is crucial for photodynamic action of dyes. Thermal deactivation and luminescence emission compete with this process, so investigation of these alternative paths of sensitizer deactivation provides information about photodynamic action. The incorporation of the investigated dyes into cells and the perturbation of the cell structure caused by the dyes, the incubation solvent and the activator were investigated by comparing the spectral properties of PBMC before and after stimulation and incubation. Incubation of the cells for 1 h in a solution of Solar Pc in 99.5% aqueous DMSO, resulted in an efficient dye incorporation which was highly selective. Solar Pc being introduced much more efficiently into stimulated cells than into resting cells.

[Microheterogeneity of two acute phase proteins in patients with ovarian carcinoma]. / Mikroheterogennosc wybranych bialek ostrej fazy u chorych z rakiem jajnika.

Concentrations of two acute phase proteins: alpha 2-macroglobulin (alpha 2M) and transferrin (Tf) as well as glycosylation profiles of alpha 2M and Tf were studied in sera samples from 13 patients suffering from ovarian cancer. In the observed group of patients with ovarian cancer in whom the progression of disease was noticed low concentrations of both investigated proteins were present. On the contrary, the microheterogeneity of both alpha 2M and Tf was changed towards variants of both proteins more weakly reacting with ConA, what was previously described for AGP and ACT in all chronic inflammatory conditions including cancer.

[Evaluation of expression of polymorphonuclear neutrophil surface receptors in patients with type 1 diabetes]. / Ocena ekspresji receptorów powierzchniowych granulocytów obojetnochlonnych u chorych na cukrzyce typu 1.

UNLABELLED: Polymorphonuclear neutrophils (PMN) play an important role in the pathogenesis of diabetic microvascular complications. Stimulation of these cells is associated with the appearance of specific receptors on their surface. The aim of the study was to evaluate the expression of the receptors specific for PMN: CD 11b, CD 18. The study was performed in a group of 23 type 1 diabetic patients, aged from 19 to 47 years (mean 30.7 +/- 8.6 years), including 15 females and 8 males (mean diabetes duration time 13.7 +/- 7.5 years; mean HbA1c 7.9 +/- 2.5%). The expression of PMN surface receptors was measured by flow cytometry using a ORTHO DIAGNOSTIC SYSTEM cytofluorimeter. The results were presented as a PMN percentage indicating expression of CD 11b and CD 18. In comparison to healthy controls, there was a significant increase in the number of PMN, both with CD 11b and CD 18 receptors present--(CD 11b: 93.2 +/- 3.4 vs 98.0 +/- 1.9% p < 0.05), (CD 18: 98.5 +/- 0.47 vs 99.4 +/- 0.7%, p < 0.05). CONCLUSION: In patients with type 1 diabetes, PMNs demonstrate a pronounced expression of surface receptors which may indicate an enhanced activity of these cells. The increase of expression of surface receptors is independent of diabetes duration time and HbA1c.

Analysis of the distribution of peripheral blood lymphocyte subsets associated with chronic ethanol treatment in rats with a disturbed circadian cycle.

The present study examined the composition of lymphoid subsets in the peripheral blood of alcohol-preferring (PRF) and non-preferring (NPF) rats, in an experimental model of alcoholism involving the disruption of the circadian cycle. The absolute and relative number of lymphocytes in specific subsets (CD3, CD4, CD8, NK, CD45RA) were measured using the flow cytometry method. When control animals with a disrupted circadian cycle (KN) were compared with a normal diurnal cycle group (KD), it was noticed that this disruption led to an increase in the absolute number of lymphocytes T (CD3(+), CD4(+) and CD8(+) cells) and lymphocytes B (CD45RA(+)). After the period of time when the alcohol preference was seen, there was a change in response - as measured by the numbers and the percentage of lymphoid subsets in NPF rats - involving a lowering of NK and CD45RA(+) cells. It seems that these animals exhibit higher sensitivity towards prolonged ethanol intoxication. However, the PRF animals - for whom the analysed values were close to those of the control group (KN) - tolerated the toxic effects of ethanol better and this may be related to their genetic predisposition.

[Monitoring of the inflammation in children before and after tonsillectomy]. / Monitorowanie stanu zapalnego u dzieci przed i po tonsylektomii.

A group of 54 children aged from three to 13 years was qualified to tonsillectomy for laryngological indications. In sera of all children following measurements were performed: the concentrations of C-reactive protein (CRP), alpha1-acid glycoprotein (AGP) and alpha1-antichymotrypsin (ACT) were measured using rocket immunoelectrophoresis according to Laurell, also concentrations of three main immunoglobulin classes (IgA, IgG, IgM) and antistreptolysin titer. The microheterogeneity of both AGP and ACT was investigated, using crossed affinity immunoelectrophoresis according to Bog-Hansen with Concanavalin A (Con A) as a ligand. Results were expressed as reactivity coefficients (RC), being the proportion of all Con A-reacting variants to the non-reacting variant. It is worth mentioning that there was no difference in all investigated parameters as well between groups obtained by categorizing children according to the presence or absence of elevated antistreptolysin titer. It may mean that at least in some cases the chronic inflammation was caused by streptococci non-producing streptolysin O. The results obtained suggest that the absence of the arthritic pain does not exclude the need of antibiotic therapy in children after tonsillectomy. Generally it is postulated that estimation of acute phase proteins concentrations and glycosylation profiles, which were previously shown to be useful in clinical assessment of various diseases may serve as additional marker in laryngology in cases where indications to tonsillectomy are still controversial.

[Prognostic significance of DNA analysis in neoplastic cells of patients with squamous cell lung neoplasms treated surgically]. / Prognostyczne znaczenie zawartosci DNA w komórkach nowotworowych u chorych na raka plaskonablonkowego pluca leczonych chirurgicznie.

The aim of our study was the assessment of the ploidia and its influence on the remission time and the survival of surgically treated patients with squamous cell lung cancer. The results were then related to the clinical staging, grading, size and localization of the tumor. The tissue samples (n = 60) of squamous cell lung carcinoma resected between 1995-1996 at the Department of Thoracic Surgery at University of Medical Sciences in Poznan were prepared using modified Hedley's method. The measurement was done in Cytoron Absolute flow cytometer. The abnormal DNA (aneuploid) was found in 40% of tumors. In two-year observation period more patients died with aneuploid tumors (54.6% deaths) than with diploid tumors (35.2% deaths). No significant correlation was found between the ploidy and frequency of metastasis to regional lymph nodes, tumor size, localization and grading. The estimation of the DNA content in the cancer cells seems to be a significant and valuable prognostic factor. The measurement of the DNA content can be useful in patients to estimate risk of recurrence after surgery.

Intense acute phase response in ischemic patients.

The aim of this study was to estimate the qualitative and quantitative changes of acute phase proteins in patients suffering from coronary heart disease. The study was carried out on 74 patients and 12 healthy volunteers. The patients were divided into three groups as follows: patients with myocardial infarction (n=37), Group I--without heart failure, Group II--with heart failure (II-III NYHA), Group III--patients with unstable angina pectoris (n=35); controls-healthy volunteers (n=12). The immunological measurements were performed at the beginning of hospitalisation (point 0), after 4, 8, 12 and 72 h, and after 6, 9 and 12 days of hospitalisation. The concentrations of C-reactive protein (CRP), alpha1-acid glycoprotein (AGP) and alpha1-antichymotrypsin (ACT) were measured using rocket immunoelectrophoresis according to Laurell. Glycosylation profiles of AGP and ACT were determined using crossed affinity immunoelectrophoresis with Con A as ligand according to Bøg-Hansen. Between Groups I and II statistically significant differences were observed for all investigated parameters. Highest concentration values were observed for Groups II and III; for Group II they appeared earlier than for Group III. The maximal values for reactivity coefficients (AGP-RC and ACT-RC) were observed earlier than the respective maximal values of concentrations. Continuous activation occurring in unstable angina leads to a more rapid increase in the concentrations of acute phase proteins and more marked alterations in their glycosylation profiles. In a way these patients seem to be 'primed' with constant stimulation, so that they respond dramatically to the stimulus of ischemia.

The incorporation of various porphyrins into blood cells measured via flow cytometry, absorption and emission spectroscopy.

The incorporation of the five following porphyrins: meso-tetra(4-phenyl)porphyrin (TPP); meso-tetra(4-sulfonato-phenyl)porphyrin (TPPS4); meso-tetra(4-naphthyl)porphyrin (TNP); tri-sulfo-tetra-phenyl porphyrin (TPPS3) and tetra-sulfonato-naphthyl porphyrin (TNPS4) into human blood cells was investigated using flow cytometry, and absorption and emission spectroscopy. The percentage of stained cells, measured in a fluorescence cytometer, provided information on the efficiency of incorporation of fluorescent dye molecules into different types of cells. The yield of the incorporation of a dye was dependent on the type of dye and the solvent used for cell incubation. The degree of dye aggregation and ionization varied with the incubation medium, but dye molecules incorporated into cells seemed to be restricted to those in the monomeric state, exhibiting similar fluorescence yield. Of the three sulfonated porphyrins investigated only TPPS4 was efficiently incorporated into leukocytes. In the incubation solvent, this dye was in monomeric and neutral form. TPPS3 which was also in monomeric form, practically was not incorporated into cells. TPP and TNP dissolved in 5% aqueous dimethyl sulfoxide were present mostly in aggregated forms but they penetrated the cells with a high efficiency.

The differences in the expression of CD45 isoforms on peripheral blood lymphocytes derived from patients with seasonal or perennial atopic allergy.

In order to determine the role of memory/naive T cells in atopic allergy patients we analyzed peripheral blood mononuclear cells before and during the grass pollen season. The study comprised 28 patients with seasonal symptoms of atopic allergy and 18 with perennial symptoms. Flow cytometry was employed to detect the expression of CD3, CD4, CD4CD45RA, CD4CD45RO, CD8, CD16, and CD19 molecules on peripheral blood lymphocytes. Allergic patients showed a decreased proportion of memory (CD4+CD45RO+) T cells compared with healthy subject (p < 0.05). The proportion of naive (CD4+CD45RA+) helper T cells did not differ between allergic patients and controls. The percentage of CD4+CD45RO+ cells increased during natural antigen exposure (grass pollen season) in allergic patients with seasonal symptoms. The results show at least two important observations. A potential homing tendency to nasal, bronchial and conjunctival mucosa of memory T cells (CD45RO) in atopic allergy patients may explain their deficiency in peripheral blood. Secondly, the grass pollen season may switch their phenotype from naive into memory T cells causing the increase of CD45RO cells. These events do not occur in non-allergic individuals and may thus constitute new insight into the basic mechanism of atopic allergy.

Lymphocyte subpopulations in the blood of women with HPV 16 positive and negative cervical cancer.

In this study the morphologic features and subpopulation of lymphocytes in the blood of women with uterine cervical cancer HPV 16 positive and HPV 16 negative was compared. The essential morphological differentiation in two groups of cancer was not found, but a statistically significant increase in CD 19 lymphocytes in the blood of women with cervical cancer HPV 16 positive was discovered. The authors suggest, that an increase in CD 19 might be connected with stronger expression of virus-oncogenic antigens on the neoplasma cells.

Influence of recipient pretreatment with donor spleen cells and 15-deoxyspergualin on rat skin graft survival.

Experimental studies performed in rat kidney transplantation showed that treatment with 15-deoxyspergualin (15-DOS) for 14 days after grafting may induce both permanent graft function and specific immune tolerance. The aim of this study was to check if donor spleen cell transfer and 15-DOS pretreatment before transplantation prolong skin graft survival. Pretreatment of the recipient with donor cells induced slight prolongation of subsequent skin graft survival. Addition of 15-DOS pretreatment for 14 days in a dose of 2.0 mg/kg b.w. to cell transfer did not influence survival of the graft. On the other hand, in the recipients receiving allogeneic spleen cells before transplantation and subtherapeutic doses of 15-DOS directly after grafting the prolongation of skin graft survival was observed. These results indicate that pretreatment of the recipients with donor cells before allogeneic skin transplantation allows for reduction of immunosuppression after grafting.