Rep-PCR genotyping of infectious Acinetobacter spp. strains from patients treated in Intensive Care Unit of Emergency Department (ICU of ED)--preliminary report.

PURPOSE: Strains of Acinetobacter spp. are responsible for a considerable percentage of hospital infections. These pathogens have colonized hospital environment and developed resistance to many currently available antibiotics. The aim of this study was one year-long analysis of the occurrence of multiresistant strains of Acinetobacter spp. in population of patients hospitalized in ICU of ED and determination of their genetic similarity. MATERIAL/METHODS: Subject of research was the population of patients admitted to ED of University Hospital in Bialystok in the period from 01.08.2010 to 01.08.2011. In the analysed group of patients, infections were identified on the basis of the guidelines of CDC. Identification and drug susceptibility of strains was specified using the automatic methods with the analyzer Vitek 2XL. Genotyping using Rep-PCR method in DiversiLab system was performed on strains of Acinetobacter spp. to determinate their genetic similarity. RESULTS: During analyzed period 405 patients were hospitalized, from 14 of them multiresistant strains of Acinetobacter spp. were isolated. Conducted genetic research allowed to detect 5 clones. Rep-PCR method in DiversiLab system enabled to learn that different clone of multiresistant strain of Acinetobacter spp. is responsible for variable forms of infection. CONCLUSIONS: Results of conducted research suggest that genotyping with rep-PCR method in DiversiLab system is useful tool in diagnostics of clones of multiresistant pathogens isolated from patients requiring intensive care, hospitalized in ED. Genotyping with rep-PCR method combined with epidemiological investigation enables to establish ways of spreading of multiresistant strains of Acinetobacter spp. in ED.

Altered balance between Th17 and Th1 cells at mucosal sites predicts AIDS progression in simian immunodeficiency virus-infected macaques.

Loss of CD4(+) T cells in the gut is necessary but not sufficient to cause AIDS in animal models, raising the possibility that a differential loss of CD4(+) T-cell subtypes may be important. We found that CD4(+) T cells that produce interleukin (IL)-17, a recently identified lineage of effector CD4(+) T-helper cells, are infected by SIV(mac251)in vitro and in vivo, and are found at lower frequency at mucosal and systemic sites within a few weeks from infection. In highly viremic animals, Th1 cells predominates over Th17 T cells and the frequency of Th17 cells at mucosal sites is negatively correlated with plasma virus level. Because Th17 cells play a central role in innate and adaptive immune response to extracellular bacteria, our finding may explain the chronic enteropathy in human immunodeficiency virus (HIV) infection. Thus, therapeutic approaches that reconstitute an adequate balance between Th1 and Th17 may be beneficial in the treatment of HIV infection.

Design and in vivo immunogenicity of a polyvalent vaccine based on SIVmac regulatory genes.

Most vaccine modalities for human immunodeficiency virus type 1 (HIV-1) tested for immunogenicity and efficacy in the SIVmac (simian immunodeficiency virus) macaque model do not include the viral regulatory proteins. Because viral regulatory proteins are expressed early during the virus life cycle and represent an additional source of antigens, their inclusion as a vaccine component may increase the overall virus-specific immune response in vaccinees. However, at least two of the early proteins, Tat and Nef, may be immunosuppressive, limiting their usefulness as components of an SIV vaccine. We have constructed a polyvalent chimeric protein in which the open reading frames for Tat and Nef have been reassorted and the nuclear localization sequence for Tat and Rev and the myristoylation site for Nef have been removed. The resulting DNA plasmid (pDNA-SIV-Retanef) (pDNA-SIV-RTN) encodes a protein of 55 kDa (Retanef) that localizes at the steady state in the cytoplasma of transfected cells. Both the DNA-SIV-RTN and the highly attenuated recombinant poxvirus vector NYVAC-SIV-RTN were demonstrated to be immunogenic in SIVmac251-infected macaques treated with ART as well as in naive macaques. An equivalent strategy may be used for the generation of polyvalent antigens encoding the regulatory proteins in a HIV-1 vaccine candidate.

ALVAC-SIV-gag-pol-env-based vaccination and macaque major histocompatibility complex class I (A*01) delay simian immunodeficiency virus SIVmac-induced immunodeficiency.

T-cell-mediated immune effector mechanisms play an important role in the containment of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) replication after infection. Both vaccination- and infection-induced T-cell responses are dependent on the host major histocompatibility complex classes I and II (MHC-I and MHC-II) antigens. Here we report that both inherent, host-dependent immune responses to SIVmac251 infection and vaccination-induced immune responses to viral antigens were able to reduce virus replication and/or CD4+ T-cell loss. Both the presence of the MHC-I Mamu-A*01 genotype and vaccination of rhesus macaques with ALVAC-SIV-gag-pol-env (ALVAC-SIV-gpe) contributed to the restriction of SIVmac251 replication during primary infection, preservation of CD4+ T cells, and delayed disease progression following intrarectal challenge exposure of the animals to SIV(mac251 (561)). ALVAC-SIV-gpe immunization induced cytotoxic T-lymphocyte (CTL) responses cumulatively in 67% of the immunized animals. Following viral challenge, a significant secondary virus-specific CD8+ T-cell response was observed in the vaccinated macaques. In the same immunized macaques, a decrease in virus load during primary infection (P = 0.0078) and protection from CD4 loss during both acute and chronic phases of infection (P = 0.0099 and P = 0.03, respectively) were observed. A trend for enhanced survival of the vaccinated macaques was also observed. Neither boosting the ALVAC-SIV-gpe with gp120 immunizations nor administering the vaccine by the combination of mucosal and systemic immunization routes increased significantly the protective effect of the ALVAC-SIV-gpe vaccine. While assessing the role of MHC-I Mamu-A*01 alone in the restriction of viremia following challenge of nonvaccinated animals with other SIV isolates, we observed that the virus load was not significantly lower in Mamu-A*01-positive macaques following intravenous challenge with either SIV(mac251 (561)) or SIV(SME660). However, a significant delay in CD4+ T-cell loss was observed in Mamu-A*01-positive macaques in each group. Of interest, in the case of intravenous or intrarectal challenge with the chimeric SIV/HIV strains SHIV(89.6P) or SHIV(KU2), respectively, MHC-I Mamu-A*01-positive macaques did not significantly restrict primary viremia. The finding of the protective effect of the Mamu-A*01 molecule parallels the protective effect of the B*5701 HLA allele in HIV-1-infected humans and needs to be accounted for in the evaluation of vaccine efficacy against SIV challenge models.

Potentiation of simian immunodeficiency virus (SIV)-specific CD4(+) and CD8(+) T cell responses by a DNA-SIV and NYVAC-SIV prime/boost regimen.

T cell-mediated immune responses play an important role in the containment of HIV-1 replication. Therefore, an effective vaccine against HIV-1 should be able to elicit high frequencies of virus-specific CD8(+) and CD4(+) T cells. The highly attenuated poxvirus-based vaccine candidate, NYVAC-SIV-gag-pol-env (NYVAC-SIV-gpe), has been shown to induce and/or expand SIV-specific CD4(+) and CD8(+) T cell responses in both naive and infected macaques. In this study, the immunogenicity of NYVAC-SIV-gpe alone was compared with a combination regimen where priming with an optimized DNA-SIV-gag-env vaccine candidate was followed by a NYVAC-SIV-gpe boost. In macaques immunized with the prime-boost regimen, the extent and durability of CD8(+) T cell response to an immunodominant SIV gag epitope was increased and these animals recognized a broader array of subdominant SIV epitopes in the cytolytic assay. In addition, the prime-boost regimen significantly enhanced the proliferative responses to both SIV gag and env proteins. Thus, the combination of these vaccine modalities may represent a valuable strategy in the development of a vaccine for HIV.

Impairment of Gag-specific CD8(+) T-cell function in mucosal and systemic compartments of simian immunodeficiency virus mac251- and simian-human immunodeficiency virus KU2-infected macaques.

The identification of several simian immunodeficiency virus mac251 (SIV(mac251)) cytotoxic T-lymphocyte epitopes recognized by CD8(+) T cells of infected rhesus macaques carrying the Mamu-A*01 molecule and the use of peptide-major histocompatibility complex tetrameric complexes enable the study of the frequency, breadth, functionality, and distribution of virus-specific CD8(+) T cells in the body. To begin to address these issues, we have performed a pilot study to measure the virus-specific CD8(+) and CD4(+) T-cell response in the blood, lymph nodes, spleen, and gastrointestinal lymphoid tissues of eight Mamu-A*01-positive macaques, six of those infected with SIV(mac251) and two infected with the pathogenic simian-human immunodeficiency virus KU2. We focused on the analysis of the response to peptide p11C, C-M (Gag 181), since it was predominant in most tissues of all macaques. Five macaques restricted viral replication effectively, whereas the remaining three failed to control viremia and experienced a progressive loss of CD4(+) T cells. The frequency of the Gag 181 (p11C, C-->M) immunodominant response varied among different tissues of the same animal and in the same tissues from different animals. We found that the functionality of this virus-specific CD8(+) T-cell population could not be assumed based on the ability to specifically bind to the Gag 181 tetramer, particularly in the mucosal tissues of some of the macaques infected by SIV(mac251) that were progressing to disease. Overall, the functionality of CD8(+) tetramer-binding T cells in tissues assessed by either measurement of cytolytic activity or the ability of these cells to produce gamma interferon or tumor necrosis factor alpha was low and was even lower in the mucosal tissue than in blood or spleen of some SIV(mac251)-infected animals that failed to control viremia. The data obtained in this pilot study lead to the hypothesis that disease progression may be associated with loss of virus-specific CD8(+) T-cell function.

Differences in time of virus appearance in the blood and virus-specific immune responses in intravenous and intrarectal primary SIVmac251 infection of rhesus macaques; a pilot study.

BACKGROUND: HIV-I can be transmitted by intravenous inoculation of contaminated blood or blood product or sexually through mucosal surfaces. Here we performed a pilot study in the SIVmac251 macaque model to address whether the route of viral entry influences the kinetics of the appearance and the size of virus-specific immune in different tissue compartments. METHODS: For this purpose, of 2 genetically defined Mamu-A*01-positive macaques, 1 was exposed intravenously and the other intrarectally to the same SIVmac251 viral stock and virus-specific CD8+ T-cells were measured within the first 12 days of infection in the blood and at day 12 in several tissues following euthanasia. RESULTS: Virus-specific CD8+ T-cell responses to Gag, Env, and particularly Tat appeared earlier in the blood of the animal exposed by the mucosal route than in the animal exposed intravenously. The magnitude of these virus-specific responses was consistently higher in the systemic tissues and GALT of the macaque exposed by the intravenous route, suggesting a higher viral burden in the tissues as reflected by the faster appearance of virus in plasma. Differences in the ability of the virus-specific CD8+ T-cells to respond in vitro to specific peptide stimulation were also observed and the greatest proliferative ability was found in the GALT of the animal infected by the intrarectal route. CONCLUSIONS: These data may suggest that the natural mucosal barrier may delay viral spreading. The consequences of this observation, if confirmed in studies with a larger number of animals, may have implications in vaccine development.

Collagenolytic activity of methylcholanthrene-induced rat fibrosarcoma.

It has been found that the methylcholanthrene-induced rat fibrosarcoma contains an enzyme (probably a cathepsin) which digests type I and type III collagens in acid pH. At physiological pH no proteolytic activity against collagen was found. It may be concluded that the tumour collagen is degraded mainly by the action of cathepsin(s).

[Proteolytic and collagenolytic activities of experimental methylcholanthrene-induced fibrosarcoma]. / Aktywnosc proteolityczna i kolagenolityczna doswiadczalnego wlókniakomiesaka indukowanego metylocholantrenem.

Rat fibrosarcoma induced by methylcholanthrene exhibited proteolytic activity against type I collagen at acidic range of pH values. This activity was not only collagen-specific as it was able to cause hemoglobin degradation to low-molecular products. No collagenolytic activity was found near physiological values pH.