Anti-Hepatitis C Virus T-Cell Immunity in the Context of Multiple Exposures to the Virus

Characterisation of Hepatitis C virus (HCV)-specific CD8⁺ T-cell responses in the context of multiple HCV exposures is critical to identify broadly protective immune responses necessary for an effective HCV vaccine against the different HCV genotypes. However, host and viral genetic diversity complicates vaccine development. To compensate for the observed variation in circulating autologous viruses and host molecules that restrict antigen presentation (human leucocyte antigens; HLA), this study used a reverse genomics approach that identified sites of viral adaptation to HLA-restricted T-cell immune pressure to predict genotype-specific HCV CD8⁺ T-cell targets. Peptides representing these putative HCV CD8⁺ T-cell targets, and their adapted form, were used in individualised IFN-γ ELISpot assays to screen for HCV-specific T-cell responses in 133 HCV-seropositive subjects with high-risk of multiple HCV exposures. The data obtained from this study i) confirmed that genetic studies of viral evolution is an effective approach to detect novel in vivo HCV T-cell targets, ii) showed that HCV-specific T-cell epitopes can be recognised in their adapted form and would not have been detected using wild-type peptides and iii) showed that HCV-specific T-cell (but not antibody) responses against alternate genotypes in chronic HCV-infected subjects are readily found, implying clearance of previous alternate genotype infection. In summary, HCV adaptation to HLA Class I-restricted T-cell responses plays a central role in anti-HCV immunity and multiple HCV genotype exposure is highly prevalent in at-risk exposure populations, which are important considerations for future vaccine design.     

On the morphology of spermatozoa of tuco-tucos, Ctenomys (Rodentia: Ctenomyidae): New data and its implications for the evolution of the genus

Sperm morphology was studied in 10 species of the caviomorph rodent Ctenomys. Ctenomys argentinus, C. conoueri, C. dorbigny and C.perrensis had symmetric spermatozoa with paddle-like heads. Ctenomys australis, C. mendocinus, C. porteousi, C. rionegrensis and Ctenomys sp., on the other hand, had spermatozoa with paddle-like heads but with the tail inserted at one side of the central axis and a nuclear caudal extension originating from the base of the head at the opposite side of the insertion of the tail and running parallel to the flagellum; these spermatozoa are referred to as simple-asymmetric. In C. yolandae, a complex-asymmetric morphological type not previously described for the genus was found. This type is characterized by the presence of two nuclear caudal extensions. Symmetric spermatozoa (total length = 52 pm) were shorter than asymmetric (both simple and complex) ones (total length = 87 pm). In spite of these differences, the relative size of heads, midpieces and tails were maintained in the three groups, representing 12%, I I YO and 88% of the average total length, respectively. Within each group of species bearing the same sperm type, a low interspecific variability both in morphological patterns and dimensions of sperm cells was observed. This low interspecific variability associated with the north to south geographical distribution of species having, respectively, symmetric and asymmetric spermatozoa, suggests that these characters appeared at an early stage in evolution of the group, and probably played an important role in the first steps of speciation by promoting reproductive isolation.     

The sex-determining zinc finger sequences in XY females of Akodon azarae (Rodentia, Cricetidae)

Wild populations of Akodon azarae comprise females with a karyotype indistinguishable from that of males. These individuals were formerly assumed to be Xx, the x being an X chromosome with a deletion of most of its long arm. By using a DNA probe derived from the testis-determining region of the human Y chromosome (comprising a candidate gene for the testis-determining factor, Y-linked zinc finger [ZFY]), we demonstrate that A. azarae gonosomally variant females are XY and not Xx. The ZFY sequences in A. azarae are amplified and located in two different families of EcoRI fragments derived from Y-chromosome DNA. No rearrangement or change in the state of methylation of ZFY or ZFX (X-linked zinc finger) sequences were found in XY females. We propose that sex reversal in A. azarae may be mediated by a gene or genes other than ZFX or ZFY.     

Inhibition of allergic inflammation in the airways using aerosolized antisense to Syk kinase

Activation of the protein tyrosine kinase Syk is an early event that follows cross-linking of Fc gamma R and Fc epsilon R, leading to the release of biologically active molecules in inflammation. We reported previously that aerosolized Syk antisense oligodeoxynucleotides (ASO) depresses Syk expression in inflammatory cells, the release of mediators from alveolar macrophages, and pulmonary inflammation. To study the effect of Syk ASO in allergic inflammation and airway hyperresponsiveness, we used the Brown Norway rat model of OVA-induced allergic asthma. Syk ASO, delivered in a liposome, carrier/lipid complex by aerosol to rats, significantly inhibited the Ag-induced inflammatory cell infiltrate in the bronchoalveolar space, decreasing both neutrophilia and eosinophilia. The number of eosinophils in the lung parenchyma was also diminished. Syk ASO also depressed up-regulation of the expression of beta(2) integrins, alpha(4) integrin, and ICAM-1 in bronchoalveolar lavage leukocytes and reversed the Ag-induced decrease in CD62L expression on neutrophils. Furthermore, the increase in TNF levels in bronchoalveolar lavage following Ag challenge was significantly inhibited. Syk ASO also suppressed Ag-mediated contraction of the trachea in a complementary model. Thus, aerosolized Syk ASO suppresses many of the central components of allergic asthma and inflammation and may provide a new therapeutic approach.     

Growth and reproduction of two species of Akodon and their hybrids

Growth and reproduction of Akodon molinae (A.m.), A. dolores (A.d.) (Rodentia: Cricetidae), and their hybrids are described. A.d. showed less fertility under animal room conditions, fewer "successful" matings, similar litter sizes, and an altered sex proportion as compared to A.m.; A.d. ovulatory index is supposed to be higher than mean litter size. X-linked lethal gene(s) and chromosome polymorphisms are possible causes of these variations. Interspecific crosses showed a marked seasonal (summer) tendency in parturitions. Intraspecific matings also produced young during winter. F1 hybrids showed a reversed tendency, while F2 matings returned to the parental pattern. A.d. male X A.m. female and F1 crosses presented larger litter sizes than the other pairings. A.m. exhibited sexual differences in weight after 60 days of age. A.d. and F1 hybrids did not show significant differences in weight of both sexes at any age. A.m. males and females were heavier than A.d. individuals except 12 months after birth. F1 hybrids born to A.m. male were heavier at birth than the A.d. female s interspecific offspring, and may be interpreted as a maternal influence of the A.m. females. Hybrids were heavier than their parents at birth and some of them were also heavier at 21 days, and probably is due to pseudoheterosis. The comparison of body measurements between sexes of both species did not give conclusive results. A.m. females were larger than females of A.d. Males of both species showed similar total lengths; A.m. males had larger body lengths and shorter tails. Body measurements were not related to weight variations. However, differences in weight and measurements between sexes appeared simultaneously at 2-6 months. This probably reflected the attainment of maturity.     

Seasonal changes in ovarian steroid hormone concentrations in the large hairy armadillo (Chaetophractus villosus) and the crying armadillo (Chaetophractus vellerosus)

Knowledge of armadillo reproductive physiology is essential for developing ex situ and in situ assisted reproductive techniques for propagating and/or controlling populations of these animals. The present study included assessment of fecal sex steroids by radioimmunoassay, determining reproductive status via monitoring ovarian activity (in the wild) and therefore reproductive status, in wild females of the large hairy armadillo (Chaetophractus villosus) and the crying armadillo (Chaetophractus vellerosus) in the southern hemisphere. Plasma and fresh fecal progesterone concentrations were not significantly correlated in either species. However, in both species, there was a significant positive correlation between plasma progesterone and dry fecal progesterone concentrations (r = 0.82, P < 0.05 and r = 0.60, P < 0.05, respectively). Dry fecal progesterone and estradiol concentrations were measured in one captive C. villosus (average baseline progesterone and estradiol concentrations 28.72 ± 11.75 ng/g dry feces and 3.04 ± 0.80 ng/g dry feces, respectively) and one captive C. vellerosus (average baseline progesterone and estradiol concentrations 14.05 ± 3.03 ng/g dry feces and 3.46 ± 1.20 ng/g dry feces, respectively) to detect hormonal peaks over 1 y; these occurred from late fall to early summer. Feces from wild C. villosus and C. vellerosus were also collected over 1 y to determine progesterone peaks, which occurred in winter and spring in both species (with no peaks during the summer or fall). Accordingly, C. villosus and C. vellerosus had a seasonal reproductive pattern. The significant correlations between dry fecal and plasma progesterone concentrations validated this method for monitoring reproductive status in these species.     

Meiotic recombination and spermatogenic impairment in Mus musculus domesticus carrying multiple simple Robertsonian translocations

We quantitatively analyzed the spermatogenic process, including evaluation of seminiferous tubules with defective cycles, rates of germ cell death and sperm morphology, in adult male mice with standard telocentric chromosomes (2n = 40, CD1 strain), homozygous (2n = 24, Mil II population) and heterozygous (2n = 24 x 40) for Robertsonian (Rb) rearrangements. The animals were analyzed at three different ages: three, five and seven months after birth. The number and position of crossover events were also determined by chiasmata counting and immunostaining with an antibody against mouse MLH1 protein. Our analysis of spermatogenesis confirms the impairment of the spermatogenic process in multiple simple heterozygotes due to both germ cell and abnormal sperm morphology. The detrimental effects exerted by Rb heterozygosities were found to be at least partially buffered with time: the frequency of defective tubules was lower and germ cell survival and sperm morphology better in 7-month-old animals than in the 3- and 5-month-old mice. While there are previously published data on germ cell death in multiple simple heterozygotes, this is the first report of a partial rescue of spermatogenesis with time. The mean frequency of MLH1 foci was lower in Rb homozygous and heterozygous mice than in mice carrying all telocentric chromosomes. The lower number of foci in Rb mice can be ascribed to a decrease in the number of multiple chiasmata and the maintenance of single chiasmata preferentially located in the terminal region of both the telocentric and metacentric chromosomes.     

Papillary endothelial hyperplasia of the orbit: report of a case highlighting a pitfall on fine needle aspiration biopsy

BACKGROUND: Papillary endothelial hyperplasia (PEH) is an unusual form of thrombus organization that occurs predominantly in the extremities, including the head and neck. However, it is rare in the orbit/ocular region. Although the histologic features of PEH have been well described, the cytologic diagnosis remains difficult. CASE: A 63-year-old man presented with a left intraorbital mass that was increasing in size and associated with paresthesia and a recent history of excision of squamous cell carcinomas (SCC) from his left cheek. Fine needle aspiration biopsy (FNAB) yielded very limited material, predominantly blood. However, 1 Papanicolaou-stained slide showed groups of atypical cells with scanty but dense cytoplasm and large, hyperchromatic nuclei with prominent nucleoli. SCC was favored, and excision was performed. Histology showed PEH within an assumed cystic lymphangioma, associated with inflammatory and fibrotic change in the compressed supraorbital nerve. CONCLUSION: To our knowledge this is the third report on the cytology of PEH and the first report of FNAB in PEH of the orbit. The case was also unusual as it was the second metachronous PEH in the patient.     

Meiotic behavior of gonosomically variant females of Akodon azarae (Rodentia, Cricetidae)

The meiotic behavior of sex chromosomes has been investigated in variant females of Akodon azarae, both in pachytene oocytes and metaphase I. In somatic cells, these females have a heteromorphic sex pair, in which the minor chromosome has been previously interpreted as a major deletion of the long arm of the X chromosome (dX). After microspreading for synaptonemal complex analysis, pachytene oocytes show two axes of very different lengths (100:17.1), which correspond to the sex chromosomes X and dX. True synapsis is abnormally restricted (43.3%) between these sex chromosomes; on the other hand, self-synapsis of both the X and dX chromosomes is frequent (60%). Single, nonsynapsed axes or axial segments are thickened. Strong chromatin condensation occurs around nonsynapsed axes or axial segments, giving many of these sex pairs an appearance similar to an XY body ("sex vesicle"). The minor gonosome axis differs from that of the Y chromosome of male meiosis, as the former is shorter (relative to the X) and has a different synaptic behavior. In 17 metaphases I from XdX variant females, only heteromorphic, end-to-end joined sex pairs were observed. These variant females differ from the variant females of the wood lemming Myopus schisticolor in several respects, but a similar mechanism seems to be prevalent in other species of the genus Akodon. Self-synapsis of unequal gonosomes in oocytes is assumed as an escape from functional deterioration, following the hypothesis put forward by others.