Anti-human leukocyte antigen antibodies are present in blood of blood donors: is therapy with blood preparations safe for graft recipients?

BACKGROUND: Blood products infusions are often administrated to graft recipients. Post-transfusion reactions of anti-human leukocyte antigen antibodies (anti-HLA) are responsible for transfusion-related acute lung injury, but cases of graft rejection after blood product infusions were recently also proven. METHODS: The aim of this study was to assess, with the use of the very sensitive Luminex technology and traditional lymphocytotoxic test, the prevalence and cytotoxic activity of anti-HLA in blood donors with different medical histories to evaluate a potential risk of post-transfusion immune complications. Data were analyzed according to different normalized background cutoffs (1.5, 2.2; and the high cutoffs-10.8 for I class and 6.9 for II class anti-HLA). RESULTS: We observed that anti-HLA may be present in 36% of donors, and even in up to 73.6% of risk groups. Significant risk factors included female sex (23.9% to 64.2% for different cutoffs) and pregnancy history (30% to 72.5%), regardless of the cutoff used in analysis, whereas sera from female donors showed lower cytotoxicity (panel reactive antibodies). Anti-HLA were also detected in men (3.7% to 37%), in donors after a transfusion (0% to 62.5%), and even with no known risk factors (3.8% to 26.9%). CONCLUSIONS: Luminex technology is a sensitive tool in anti-HLA detection, but consensus in measurement interpretation for blood donors is needed. Selection of blood products on the basis of medical history can be a useful alternative for routine testing of blood donors. The clinical significance of treatment of graft recipients with blood products requires further study; until then, more attention should be paid to possible complications.

Analysis of specificity of anti-human leukocyte antigen antibodies in kidney recipients in reference to clinical outcome.

BACKGROUND: Anti-human leukocyte antigens antibodies (HLA) are not always the main cause of graft injury but can be a marker of immune response to the graft. The aim of this study was to analyze anti-HLA specificities with the use of the most sensitive detection method (Luminex) in reference to clinical condition. METHODS: Sera of 65 kidney recipients (n = 443) were screened with the use of the mixed LABScreen kit, and, for 47 recipients, sera with maximal normalized background ratio (NBG) were subjected to specificity testing. NBG, numbers of specificities, donor-specific antibodies (DSA), and normalized mean fluorescence index (nMFI) of DSA and maximal anti-HLA were analyzed in reference to clinical (acute rejection [AR] diagnosis, immunosuppression), histopathological (C4d staining, chronic allograft nephropathy, AR type), and laboratory parameters (creatinine). RESULTS: We observed 1 to 51 specificities, class I DSA in 26.7%, class II in 10%, and estimated DQ-DSA in 63.3% of tested patients. Patients with AR and humoral AR had significantly higher NBG, number of anti-HLA class I, DQ and DQ-DSA types, and more frequently had anti-HLA and class II DSA-positive sera (P < .052). C4d staining was associated with higher anti-HLA class I (P = .053) and class I DSA (P = .002) type numbers, and maximal anti-HLA nMFI (P = .036) and was more frequent in AR (P = .048) and class II DSA positive patients (P = .046). Patients with chronic allograft nephropathy showed higher DQ-DSA-nMFI (P = .036). DQ-DSA-nMFI and maximal anti-HLA-nMFI correlated with creatinine increase (Spearman range [SR] = 0.64, SR = 0.41). Together with NBG, maximal class I and class II anti-HLA-nMFI correlated with the number of transplantation and maximal panel-reactive antibodies ratio (SR = 0.19-0.40). CONCLUSIONS: Anti-HLA detection allows for humoral AR diagnosis but also for identification of patients with risk of any rejection. However, clear rules of anti-HLA interpretation and studies on their clinical impact are needed.

Cytokine response in patients with chronic infections caused by Staphylococcus aureus strains and diversification of their Agr system classes.

This study aimed to describe the levels of circulating cytokine levels produced by Th lymphocytes (IFN-γ, IL-4, IL-10, IL-17A), as well as the levels of cytokines produced by monocytes/macrophages (TNF-α, IL-1ß, IL-12), in patients with chronic infections caused by Staphylococcus aureus strains, particularly in the context of the diversification of their Agr system classes. The studies were conducted on adult patients, including 50 patients with chronic suppurative dermatitis, 40 patients with chronic infections of the upper respiratory tract and 25 healthy individuals (control group). Blood serum cytokine levels were measured by enzyme-linked immunosorbent assay (ELISA). S. aureus was detected in cultures of suppurative dermal exudates or of pharyngeal smears. Classes of Agr systems in the S. aureus strains were identified using polymerase chain reaction (PCR). In both groups of patients, on average, levels of IFN-γ were doubled, while levels of IL-17A were increased by 2.5-fold, which, however, was not accompanied by increased levels of TNF-α or IL-12. The data indicate that the development of S. aureus infection among the studied patients was linked to an impoverished cytokine response of monocytes/macrophages, while that induced by the pathogen lymphocytes Th17/Th1 may be responsible for promotion of the chronic inflammatory response. In parallel, no quantitative or qualitative differences were disclosed between cytokine responses manifested by subgroups of patients infected with S. aureus strains belonging to class IV Agr, as compared to patients infected with strains of classes I to III Agr. Nevertheless, in the patients, strains belonging to class IV Agr prevailed, which points to the preferential relationship between the class and the pathogenicity of S. aureus.

Lactobacillus rhamnosus administration causes sepsis in a cardiosurgical patient--is the time right to revise probiotic safety guidelines?

A 24-year-old female patient developed sepsis resulting from preoperative administration of probiotics following an aortic valve replacement. Blood cultures revealed the causative agent to be the probiotic Lactobacillus rhamnosus, which has recently been implicated as an emerging aetiology of infection in those taking probiotics. In the past few years, probiotic use in hospitals has increased greatly. However, there is growing global evidence that the use of probiotics in patients with organ failure, immunocompromised status and dysfunctional gut barrier mechanisms can cause infections. This and other reports show the importance of establishing generally recognized safety guidelines.

Antibody responses in furunculosis patients vaccinated with autologous formalin-killed Staphylococcus aureus.

Autologous vaccines (short: autovaccines) have been used since the beginning of the 20th century to treat chronic staphylococcal infections, but their mechanisms of action are still obscure. This prospective pilot study involved four patients with furunculosis who were vaccinated with autologous formalin-killed Staphylococcus aureus cells. Vaccines were individually prepared from the infecting S. aureus strain and repeatedly injected subcutaneously in increasing doses over several months. We characterized the virulence gene repertoire and spa genotype of the infecting and colonising S. aureus strains. Serum antibody responses to secreted and surface-bound bacterial antigens were determined by two-dimensional immunoblotting and flow-cytometry based assays (Luminex). All patients reported clinical improvement. Molecular characterization showed that all strains isolated from one patient over time belonged to the same S. aureus clone. Already before treatment, there was robust antibody binding to a broad range of staphylococcal antigens. Autovaccination moderately boosted the IgG response to extracellular antigens in two patients, while the antibody response of the other two patients was not affected. Similarly, vaccination moderately enhanced the antibody response against some staphylococcal surface proteins, e.g. ClfA, ClfB, SdrD and SdrE. In summary, autovaccination only slightly boosted the pre-existing serum antibody response, predominantly to bacterial surface antigens.

Association of allograft inflammatory factor-1 gene polymorphism with rheumatoid arthritis.

Human allograft inflammatory factor-1 (AIF-1) is a cytoplasmic protein primarily identified in human and rat allografts, and data from several studies suggest an important role for AIF-1 in inflammatory processes. The aim of this study was to examine the association between AIF1 rs2269475:C>T polymorphism and rheumatoid arthritis (RA). AIF1 genotype was determined by means of the polymerase chain reaction-restriction fragment length polymorphism method in 276 White patients with RA and 236 healthy subjects. The frequency of the AIF1 rs2269475 TT genotype was significantly higher in the patients with RA than in the controls (OR=5.59, 95% CI: 1.22-25.55). The frequency of T allele carriers in the patient group with RA was 31.9% vs 19.1% among controls (P=0.0003). Moreover, the frequency of individuals positive for anti-cyclic citrullinated peptide (anti-CCP) antibodies was significantly elevated in the T allele carriers (OR=8.82, 95% CI: 2.06-37.7). It is noteworthy that no significant linkage disequilibria between the AIF1 C/T and DRB1 alleles associated with RA development and anti-CCP antibody production [including the most frequent, i.e. *04 (32.7%) and *01 (23.5%)] (P>0.1) were found. Our results show that the AIF1 rs2269475 T allele is associated with increased risk of RA development. Moreover, the frequency of individuals positive for anti-CCP antibodies is significantly increased among T allele carriers.

Resistance, serotype and genetic diversity of Streptococcus pneumoniae-resistant strains isolated in the West Pomerania region of Poland in the years 2001-2005.

The aim of this study was to analyse the resistance patterns, serotypes and genetic diversity of Streptococcus pneumoniae-resistant strains isolated in the West Pomerania region of Poland. They were clinical isolates obtained during a 5-year study (2001-2005) mainly from ambulatory patients with upper respiratory tract infections. The strains showed resistance to 8 out of 9 tested antibiotics (except vancomycin) and 53.8% of the strains were multidrug-resistant (MDR). The increase over time in the number of MDR strains and in resistance degrees was not statistically significant. Resistance to cotrimoxazole was the most frequent (86.7%). Penicillin nonsusceptibility was shown in 38% of the strains and resistance to macrolides in 36.7% of the strains, mainly of MLS(B) phenotype (94.1%). A significant resistance increase was only observed for beta-lactam antibiotic. The population of S. pneumoniae-resistant strains in our region presented 31 resistance patterns, 13 serotypes and a high genetic diversity-70 pulse field gel electrophoresis (PFGE) profiles have been described: 44 of them were unique and 26 clusters consisted of 2 to 30 strains similar by more than 87%. Cluster I, grouping 30 strains of similar resistant patterns (TSH: 70%, SH, TH, T, H, S) and mainly serotype 19F, isolated over the 5 years of the study, could represent a new national clone. The polysaccharide 23-valent vaccine covers 83.5%, while the conjugated 7-, 9- and 11-valent vaccines cover 79.1-79.7% of the resistant strains collected in our region. A statistically significant decrease of vaccine coverage in time has been noted.

The association between past Chlamydia pneumoniae infection and markers of chronic inflammation in obese women.

It has been widely accepted that obesity is associated with chronic, low-grade inflammation that affects the adipose tissue as well as the entire system. The aim of this study was to assess whether past Chlamydia pneumoniae infection influences obesity phenotypes and serum levels of low-grade inflammation markers in obese, healthy premenopausal women. The study was performed on 48 obese and 42 normal-weight women, aged 31.2 +/- 7.2 years. Serum levels of C-reactive protein (CRP), tumor necrosis factor alpha (TNFalpha) and its soluble receptor R2 (sTNF-R2), and interleukin 6 (IL-6) were measured. Body composition was assessed by bioimpendance. Insulin sensitivity was assessed by quantitative insulin sensitivity check index (QUICKI). The seroprevalence of C. pneumoniae infection was 69.1% and was similar in obese and normal-weight women (75.2% and 61.9%, respectively; P = 0.18). Obese women had higher CRP than healthy controls (P < 0.05). IL-6, TNFalpha, and sTNF-R2 showed no significant differences when comparing obese and normal-weight or C. pneumoniae infected and uninfected women. In multivariate regression analysis, fat mass (P < 0.001) and QUICKI (P < 0.01), accounting for 35% of the variance of CRP and C. pneumoniae infection, did not significantly contribute to this model (P = 0.51). In conclusion, past C. pneumoniae infection was not associated with changes in chronic inflammation markers in premenopausal obese women.

Clonal distribution of superantigen genes in clinical Staphylococcus aureus isolates.

Staphylococcus aureus is both a successful human commensal and a major pathogen. The elucidation of the molecular determinants of virulence, in particular assessment of the contributions of the genetic background versus those of mobile genetic elements (MGEs), has proved difficult in this variable species. To address this, we simultaneously determined the genetic backgrounds (spa typing) and the distributions of all 19 known superantigens and the exfoliative toxins A and D (multiplex PCR) as markers for MGEs. Methicillin- sensitive S. aureus strains from Pomerania, 107 nasal and 88 blood culture isolates, were investigated. All superantigen-encoding MGEs were linked more or less tightly to the genetic background. Thus, each S. aureus clonal complex was characterized by a typical repertoire of superantigen and exfoliative toxin genes. However, within each S. aureus clonal complex and even within the same spa type, virulence gene profiles varied remarkably. Therefore, virulence genes of nasal and blood culture isolates were separately compared in each clonal complex. The results indicated a role in infection for the MGE harboring the exfoliative toxin D gene. In contrast, there was no association of superantigen genes with bloodstream invasion. In summary, we show here that the simultaneous assessment of virulence gene profiles and the genetic background increases the discriminatory power of genetic investigations into the mechanisms of S. aureus pathogenesis.

Clinical forms of infections in neonates hospitalized in clinic of obstetrics and perinatology within the space of one year.

Because of their specificity, infections in neonatal units form one of the main clinical problems. Our research involved all neonates (1019) hospitalized in Clinic of Obstetrics and Perinatology within the space of one year. Clinically manifested infections were diagnosed in the total number of 47 (4.6%) newborns, including 23 (2.4%) neonates from the neonatal unit (NU) and 24 (46.2%)--from the Neonatal Intensive Care Unit (NICU). In both units, the most commonly observed were general infections (59.6%) and pneumonias (21.3%); cerebrospinal meningitis and necrotic enteritis were diagnosed in a few cases. Urinary system infections were only found in neonates hospitalized in the NU (30.5%). The course of infection was mild in most cases.

Clinical and microbiological characteristics of hospital infections in the neonatal intensive care unit.

Neonates hospitalized in intensive care units, are exposed to a higher risk of infectious complications. The research involved 52 neonates hospitalized in the Neonatal Intensive Care Unit (NICU), Chair and Clinic of Obstetrics and Perinatology over a span of one year. The incidence of hospital infections as well as etiological factors were analyzed. Clinically manifested hospital infections were diagnosed in 38.5% of babies with very low or extremely low birth weight, in boys twice as often as in girls. Generalised invasive infections prevailed; in most cases they were caused by Gram-negative rods, mainly Klebsiella spp.

Soluble HLA class I molecules exert differentiated influence on renal graft condition.

The function of soluble HLA (sHLA) antigens in the process of immunoregulation and especially in graft tolerance versus rejection has not yet been established. It has been suggested that donor-derived sHLA may exert an immunotolerant influence on the graft. We sought to determine the role of sHLA class I in kidney graft survival by evaluating the influence of these molecules on allotypic lymphocytotoxic antibodies and the concentration of gamma interferon (INF-gamma). Analysis of sHLA was performed indirectly utilizing their ability to inhibit lymphocytotoxic reaction dependent on complement activation. To demonstrate the inhibitory properties of sHLA, we modified the NIH microcytotoxic test. Furthermore, we determined the concentration of INF-gamma in all sera samples for comparison with the intensity of the cytotoxic test. The comparison of the intensity of cytotoxic test inhibition with the concentration of INF-gamma revealed that high concentrations of this cytokine were associated with stronger inhibition of the cytotoxic test, thus with higher concentrations of sHLA class I molecules in recipient sera. We observed that high concentrations of sHLA class I molecules in recipient sera significantly inhibited cytotoxic reactions, which could contribute to a protective influence of sHLA on renal grafts. On the other hand, the observed increase of INF-gamma concentration might be caused by sHLA themselves, which would produce a detrimental influence on a transplanted organ. Therefore we concluded that the role of sHLA class I molecules in renal graft condition remains ambiguous.

The influence of angiotensin-converting enzyme gene of donor and recipient on the function of transplanted kidney.

One of the genes that is supposed to influence renal graft function is the one encoding angiotensin I-converting enzyme (ACE). It shows polymorphism in the presence (I allele) or absence (D allele) of a 287-base pair fragment. The question arises whether ACE gene polymorphism of the recipient and donor influences renal graft survival. This prospective study included 94 recipients who underwent ACE genotyping (DD, DI, II) and measured their creatinine clearance after cimetidine administration. These factors were correlated with the occurrence of acute or chronic rejection and of pharmacologic treatment of hypertension. In 27 recipients it was possible to obtain the ACE genotype of the donor. Among the recipients, 36 proved to be DD genotype, 38 ID, and 20 II. Among the donors, 10 proved to be DD genotype, 10 ID, and 7 II. The changes in creatinine clearance after cimetidine administration were not significantly different among any of the genotype subgroups. Significantly higher creatinine concentrations were found among recipients with II genotype compared to the combined group of ID and DD among patients not treated with ACE inhibitors, but not among those receiving ACE I after kidney transplantation. No differences were found in the frequency of rejection episodes among the subgroups with different ACE genotypes. No significant influence of donor ACE genotype on renal graft function was observed. In summary, the I/D genotype was not an independent prognostic factor for renal graft survival in the first 4 years after transplantation. Possibly the use of ACE I alters the influence of genotype on some parameters.

Antibiotic sensitivity of bacteria isolated in 1998 and 1999 from patients with infections of upper airways residing in Western Pomerania.

Sensitivity to antibiotics of the most common pathogens isolated from the upper airways in north-west part of Poland shown significant regional variation. The rise in resistance to penicillin for Streptococcus pneumoniae (to 22%) and to macrolides for Streptococcus pyogenes, Streptococcus pneumoniae and Staphylococcus aureus was observed. No differences in sensitivity have been found between pathogens isolated from hospital and ambulatory patients.

[Analysis of HLA class I and II antigens composition in high-risk pregnancy]. / Analiza wystepowania antygenów HLA klasy I i II w ciazy wysokiego ryzyka.

DESIGN: We have analyzed the frequency of HLA class I and II antigens in high-risk pregnancy. MATERIAL AND METHODS: Altogether, 22 gravida hospitalized at the Department of Pathology of Pregnancy and Labour, Pomeranian Medical University in Szczecin formed group I (PE) with 12 cases of preeclampsia and group II with 10 cases of the antiphospholipid syndrome (APS). The control group included 40 multigravida. Typing of HLA class I and II antigens was done using the two-stage microcytotoxic test (NIH) of Mittal. RESULTS: Antigen B35 was found more frequent in preeclampsia and antiphospholipid syndrome groups than in multigravida. Furthermore, a statistically significant difference in the frequency of homozygotes for the HLA-DR locus was noted between groups PE and APS on one hand, and controls on the other. CONCLUSIONS: Identical HLA-DR3, DR4 and DR5 antigens were found more frequently in preeclampsia, while identical DR4 and DR6 in the antiphospholipid syndrome.

[Methicillin resistant clinical strains of Staphylococcus aureus isolated from patients in the State Clinical Hospital Nr 2 in Szczecin]. / Oporne na metycyline szczepy Staphylococcus aureus izolowane od chorych w Panstwowym Szpitalu Klinicznym Nr 2 w Szczecinie.

Over a fivefold increase, from 11% to 58%, in the prevalence of methicillin-resistance was observed in 1994-95 amongst clinical isolates of Staphylococcus aureus in the State Clinical Hospital No 2 in Szczecin, one of the largest hospitals in the West Pomeranian region of Poland. The aim of this study was to see if any one particular strain was responsible for this apparent outbreak. Fifty-six randomly selected isolates were typed by SmaI macrorestriction analysis using PFGE and by analysis of antimicrobial susceptibility patterns. Results indicate the presence of two epidemic multi-drug resistant MRSA strains. Over 85% of typed MRSA belonged to the first strain, which was probably present in the hospital long before 1994. MRSA of this strain were isolated from patients in 8 hospital wards. The second strain was introduced into two wards of the hospital in the last year of the study.

Short-term storage of human haematopoietic cells. Influence of air and deoxyribonuclease I.

The aim of this study was to optimize strategy for ex vivo short-term storage of human cord blood and bone marrow haematopoietic cells. We report that the presence of air in the vials (1/2 of their volume), in which hematopoietic cells are stored, improves the survival of clonogenic progenitors. We observed that presence of air prevented a rapid decrease in the pH of storage medium. Similarly, a beneficial effect on cell survival and recovery also had an addition of Deoxyribonuclease I (DNase I). We observed that DNase I efficiently prevented cell clumping, and moreover, did not affect the clonogenecity of the haematopoietic progenitors. Therefore containers, in which haematopoietic cells are stored, should contain enough air (source of oxygen) and the storage medium itself should be supplemented with DNase I.

[Drug resistance and proticinogenic types of Proteus mirabilis isolated from urinary tract infections]. / Lekoopornosc oraz typy proticynogenne szczepów Proteus mirabilis izolowanych z zakazen dróg moczowych.

Proteus mirabilis strains (88 isolates) from hospitalised patients with urinary tract infection were tested for antibiotic susceptibility, ESBL production and their ability to produce proticin or on their susceptibility to proticin. Antibiotic susceptibility test was performed by standard disc diffusion method according to NCCLS. Proticin typing was made by the standard strain set from the B. W. Senior collection. Most (59%) strains belonged to ESBL producers and were more resistant to antibiotics than ESBL-negative strains. Predominant proticin patterns among the ESBL (+) strains were: P1,2(6)/SO (23%), P1,2/SO (13.5%). Among the ESBL-negative strains more frequent were P6/SO (16.6%), P1,2/SO (13.8%) and P3,6/SO (13.8%) proticin types.

[Bacterial flora of acne lesions in diagnostic material of the Microbiology and Immunology Department, Pomeranian Medical Academy (PAM) in Szczecin]. / Flora bakteryjna zmian tradzikowych w diagnostycznym materiale Zaklad Mikrobiologii i Immunologii PAM w Szczecinie.

A retrospective analysis of the bacterial flora of acne lesions was carried out in 320 patients. Atypical flora was isolated very rarely. The pathogenic flora was found most frequently in the Autovaccine Laboratory when the microbiological material was taken directly onto plates and the investigation was repeated several times. Atypical flora was isolated only from three cases. Propionibacterium and Staphylococcus epidermidis were found to be sensitive to the antibiotics routinely used in the treatment of acne. The highest antibacterial effect in vitro against both these species was demonstrated using rifampicin and tetracycline.