Decreased pH does not alter metamorphosis but compromises juvenile calcification of the tube worm Hydroides elegans.

Using CO2 perturbation experiments, we examined the pre- and post-settlement growth responses of a dominant biofouling tubeworm (Hydroides elegans) to a range of pH. In three different experiments, embryos were reared to, or past, metamorphosis in seawater equilibrated to CO2 values of about 480 (control), 980, 1,480, and 2,300 µatm resulting in pH values of around 8.1 (control), 7.9, 7.7, and 7.5, respectively. These three decreased pH conditions did not affect either embryo or larval development, but both larval calcification at the time of metamorphosis and early juvenile growth were adversely affected. During the 24-h settlement assay experiment, half of the metamorphosed larvae were unable to calcify tubes at pH 7.9 while almost no tubes were calcified at pH 7.7. Decreased ability to calcify at decreased pH may indicate that these calcifying tubeworms may be one of the highly threatened species in the future ocean.

Response of larval barnacle proteome to CO(2)-driven seawater acidification.

The majority of benthic marine invertebrates have a complex life cycle, during which the pelagic larvae select a suitable substrate, attach to it, and then metamorphose into benthic adults. Anthropogenic ocean acidification (OA) is postulated to affect larval metamorphic success through an altered protein expression pattern (proteome structure) and post-translational modifications. To test this hypothesis, larvae of an economically and ecologically important barnacle species Balanus amphitrite, were cultured from nauplius to the cyprid stage in the present (control) and in the projected elevated concentrations of CO(2) for the year 2100 (the OA treatment). Cyprid response to OA was analyzed at the total proteome level as well as two protein post-translational modification (phosphorylation and glycosylation) levels using a 2-DE based proteomic approach. The cyprid proteome showed OA-driven changes. Proteins that were differentially up or down regulated by OA come from three major groups, namely those related to energy-metabolism, respiration, and molecular chaperones, illustrating a potential strategy that the barnacle larvae may employ to tolerate OA stress. The differentially expressed proteins were tentatively identified as OA-responsive, effectively creating unique protein expression signatures for OA scenario of 2100. This study showed the promise of using a sentinel and non-model species to examine the impact of OA at the proteome level.

Primary chondroid melanoma.

BACKGROUND: Malignant melanoma is notorious for the wide range of histologic patterns it can assume, among the least frequent of which is chondroid melamona. METHODS: Two cases of primary chondroid melanoma of the distal lower extremity were studied. Tissue for light microscopy was fixed in formalin, embedded in paraffin, and processed routinely. In one case, transmission electron microscopy and immunohistochemical evaluation were performed. RESULTS: Both cases exhibited melanoma in-situ, a conventional (non-chondroid) invasive component, and areas of chondroid differentiation, as confirmed by strongly positive staining with Alcian blue at pH 2.5 and Safranin O. Immunohistochemically, one case expressed S-100 protein and vimentin, and did not express gp100 (HMB-45), tyrosinase, MART-1, the Mel-5 antigen, the NKI/C3 antigen, CD45Ro, cytokeratin, or desmin. Electron microscopy of the chondroid component revealed occasional tumor cells with rare, membrane-bound, electron-dense organelles; the extracellular compartment showed amorphous ground substance consistent with cartilaginous differentiation. CONCLUSIONS: Chondroid change in the absence of osteogenic differentiation is extremely rare in malignant melanoma. Melanoma should be considered in the differential diagnosis of primary cutaneous neoplasms exhibiting cartilaginous differentiation.

Utility of fine-needle aspiration in the diagnosis of recurrent pulmonary lymphangioleiomyomatosis: a case report.

Pulmonary lymphangioleiomyomatosis (PLAM) is a rare disease of undetermined cause that almost exclusively occurs in women of child-bearing age and is characterized by distinct clinical, radiologic, and pathologic findings. PLAM is usually fatal unless treated with hormonal manipulation or lung transplantation. Primary diagnosis usually requires open lung biopsy to demonstrate the characteristic hamartomatous smooth muscle proliferation. To date, cytologic diagnosis of LAM has been limited to an ancillary role. Herein, we report the diagnosis of this neoplasm in a recurrence in an abdominal lymph node using fine-needle aspiration.

The cervico-ocular reflex of normal human subjects in response to transient and sinusoidal trunk rotations.

We used the magnetic search coil technique to measure the horizontal cervico-ocular reflex (COR) of 8 subjects in response to transient or sinusoidal (0.1-1.0 Hz) trunk rotations while their heads were firmly immobilized. Although we were able to resolve eye rotations of < 0.05 degrees, the COR was hardly measurable (gain was always < 0.07). This finding, made with the most precise measurement technique used to date, suggests that the COR makes a negligible contribution to the stability of gaze in normal subjects during natural activities.

Immunization-induced decrease of the CD4+:CD8+ ratio in cats experimentally infected with feline immunodeficiency virus.

In a previous experiment a group of 15 specified pathogen free (SPF) cats were experimentally infected with a Swiss isolate of feline immunodeficiency virus (FIV). A group of 15 SPF cats served as FIV negative controls. Nine cats of each group were vaccinated with a recombinant feline leukemia virus (FeLV) vaccine, six cats in each group with a placebo vaccine. All vaccinated cats developed high antibody titers to FeLV and were protected against subsequent FeLV challenge infection. In both control groups five of six cats became persistently infected with FeLV. Unexpectedly, the primary immune response to the vaccine antigen was significantly higher in the FIV positive group than in the FIV negative. The secondary response was stronger in the FIV negative cats. The goal of the present investigation was to further study the immune response in these 30 cats. They were immunized twice with the synthetic peptide L-tyrosine-L-glutamic acid-poly(DL-alanine)-poly(L-lysine) (TGAL) 21 days apart. Blood samples were collected on four occasions during the immunization process. They were tested for antibodies to TGAL, complete blood cell counts and CD4+, CD8+ and pan-T-lymphocyte counts. The following observations were made: (1) in contrast to the FeLV vaccine experiment, the primary immune response to TGAL was not significantly stronger in the FIV positive cats when tested by enzyme-linked immunosorbent assay (2). The absolute size of the CD4+ lymphocyte population was distinctly smaller in the FIV positive than in the FIV negative cats. The lowest CD4+ values were found in the dually FIV/FeLV infected cats. (3) A population of CD8+ lymphocytes was identified that was characterized by a distinctly weaker fluorescence. The size of this population increased in FIV positive and decreased in FIV negative cats during the TGAL immunization experiment. (4) The CD4+:CD8+ ratio increased in FIV negative cats during TGAL immunization from 1.9 to 2.3. In contrast, in FIV positive animals the CD4+:CD8+ ratio decreased significantly from 1.9 to 1.3 during the same period. From these and earlier data it was concluded that in short-term FIV infection the immune response to T-cell dependent antigens may be increased over that of the controls. Immune suppression develops gradually with duration of the infection. The significant drop of the CD4+:CD8+ ratio over a 5 week immunization period suggests that antigenic stimulation may accelerate the development of immune suppression in FIV positive cats. If this is a general feature, FIV infection may provide a particularly interesting model for studying the pathogenesis of AIDS.

Characterization of a feline T-cell-specific monoclonal antibody reactive with a CD5-like molecule.

The 43 monoclonal antibody raised against feline T cells was found to react with a single-chain glycoprotein of Mr 72,000 that is present on most thymocytes, 60% of lymph node cells, 20% of splenocytes, and 45% of blood mononuclear cells. All CD4+ and CD8+ T cells were found to express the 43-reactive determinant, as did a small subpopulation of CD4-/CD8-/IgM- lymphocytes in the periphery. The 43-reactive determinant was not detected on B cells, macrophages, or other types of blood cells. The 43 antigen was phosphorylated in resting and activated T cells. Its expression was upregulated by stimulation with phorbol myristate acetate and with phytohemagglutinin. When added to concanavalin A-stimulated T-cell cultures in low concentrations, the 43 antibody was found to augment mitogenesis. The data indicate that this antibody may identify a CD5 homologue on feline T cells.

Decline in CD4+ cell numbers in cats with naturally acquired feline immunodeficiency virus infection.

T-cell subsets were studied by fluorescence-activated cell sorter analysis in 57 feline immunodeficiency virus (FIV)-seropositive cats with naturally acquired FIV infection to see whether CD4(+)-CD8+ alterations were comparable to those observed in human immunodeficiency virus-infected patients. CD4+ values were decreased and CD8+ values were increased. The CD4+/CD8+ ratio was reduced to 1.6, compared with 3.3 in 33 FIV-seronegative control cats. Variance analysis of data showed a significant influence of FIV seropositivity, sex, and spaying of female cats on CD4+ values. CD8+ values were significantly influenced by FIV seropositivity, age, and breed. These findings indicate a similarity between FIV and human immunodeficiency virus infections, as far as alterations of T-cell subsets are concerned.

[T-helper and T-suppressor lymphocyte subpopulations in the peripheral blood of spontaneously FIV-positive cats]. / T-Helfer- und T-Suppressor-Lymphozyten-Subpopulationen im peripheren Blut spontan FIV-positiver Katzen.

Reduced CD4(+)-T-helper-lymphocyte- and increased CD8(+)-T-suppressor-lymphocyte-subsets were found in peripheral blood of 47 FIV-seropositive cats with naturally acquired FIV-infection. The CD4+/CD8+ ratio was decreased, too. Variance analysis of data included the variables reaction in FIV-test, age group, and race. Similarities with HIV-infection were discussed.

Experimental vaccine protection against feline immunodeficiency virus.

Infection of domestic cats with the feline immunodeficiency virus (FIV) represents an important veterinary health problem and a useful animal model for the development of vaccines against acquired immunodeficiency syndrome (AIDS). Two experimental FIV vaccines have been developed; one consisting of fixed infected cells (Vaccine 1), the other of inactivated whole virus (Vaccine 2). After 4-6 immunizations over 2-5 months, both vaccines induced a strong FIV-specific immune response including neutralizing antibody and T-cell proliferation. Vaccine 1 protected 6 of 9 and Vaccine 2 protected 5 of 6 recipient cats against any detectable infection with a low dose (10 animal ID50) of FIV given intraperitoneally 2 weeks after the final boost. One additional cat in each vaccine group had a transient infection at 5-7 weeks postchallenge following which virus could no longer be detected. Thus, a total of 13 of 15 vaccinated cats were protected against persistent infection. By contrast, 13 of 13 controls were persistently infected by this challenge. The infected cell vaccine failed to protect against a higher dose (5 x 10(4) ID50) of FIV. These results indicate that vaccine prophylaxis against natural FIV infection should be achievable and enhance optimism of the prospect of developing an effective AIDS vaccine for humans.

Flow cytometric analysis of T-lymphocyte subsets in cats.

We report a rapid, reliable method for the immunophenotype analysis of feline lymphocytes. Fluorescein isothiocyanate (FITC) conjugated to murine monoclonal antibodies f43, Fel 7 and fCD8 was used to identify phenotypes corresponding to feline T-cells, CD4+ T cells and CD8+ T cells. For isolation of white blood cells, whole blood lysis was faster, less variable and required much less sample than density gradient separation. To identify feline CD4+ and CD8+ cells simultaneously, directly conjugated FITC-fCD8 and phycoerythrin (PE) fCD4 (Fel 7) were used in two-color analysis. The two T cell sub-populations were non-overlapping. Dual-label and single-label values were not significantly different. Mean lymphocyte subset percentages in conventional and specific-pathogen-free (SPF) cats did not differ significantly. These values were: pan T lymphocytes (f43), 54.8%, CD4+ cells (Fel 7), 33.9%, and CD8+ cells (fCD8), 19.1%. Mean CD4/CD8 ratio was 1.9 in normal cats; the range was 1.2-2.6.

Development of IL-2-independent feline lymphoid cell lines chronically infected with feline immunodeficiency virus: importance for diagnostic reagents and vaccines.

Two interleukin 2 (IL-2)-independent feline immunodeficiency virus (FIV) producer cell lines (FL-4 and FL-6) were produced by selecting cells from an IL-2-dependent culture of mixed peripheral blood lymphocytes infected with FIV. The new cell lines have been stable for over 1 year and spontaneously produce FIV with an average reverse transcriptase titer of 300,000 cpm/ml and an average sucrose gradient purified viral protein concentration of 1 mg/l. FIV produced from these cultures is highly infectious in vitro and in vivo. The FL-6 cell line was phenotyped as expressing the feline CD8 and Pan-T antigens, while the FL-4 cell line expressed the CD4, CD8, and Pan-T antigens. Both cell lines, however, express high levels of viral core and envelope proteins. Paraformaldehyde-inactivated whole virus and similarly inactivated whole-cell virus preparations induced a strong antibody response to core and envelope antigens in immunized cats. The establishment of FIV-producing feline IL-2-independent peripheral blood lymphocyte lines should be valuable for the development of FIV-diagnostic reagents and vaccines and also as a model for human acquired immunodeficiency syndrome vaccine development.

Acquired immune dysfunction in cats with experimentally induced feline immunodeficiency virus infection: comparison of short-term and long-term infections.

Specific pathogen-free domestic cats with experimentally induced feline immunodeficiency virus (FIV) infections of short duration (less than or equal to 10 months) exhibited depressed total leukocyte and neutrophil numbers and a marginally decreased lymphocyte proliferative response to pokeweed mitogen (PWM), while cats with infections of more lengthy duration (greater than or equal to 25 months) exhibited normal leukocyte and neutrophil numbers but a dramatic loss of responsiveness to both PWM and concanavalin A (Con A). Cats with short-term infections exhibited a decrease in the percentage of CD4+ lymphocytes in peripheral blood and a corresponding depression of the CD4+:CD8+ ratio. Cats with long-term infections exhibited a similar but more profound perturbation of the CD4+ lymphocyte subset that also included a decrease in the absolute number of CD4+ cells. The decreased responsiveness to Con A and PWM in cats infected long term paralleled the decline in CD4+ cell counts, and the duration of infection was directly correlated with the decrease in the percentage of CD4+ cells. These data provide evidence supporting the hypothesis that FIV is the cause of an immune dysfunction in cats, with distinct similarities to that produced by human immunodeficiency virus (HIV) in people.

Lymphocyte subset alterations and viral determinants of immunodeficiency disease induction by the feline leukemia virus FeLV-FAIDS.

The FeLV-FAIDS strain of feline leukemia virus consistently induces fatal immunodeficiency. To investigate the immunopathogenesis and viral genetic determinants responsible for the induction of immunodeficiency disease in vivo, we have generated chimeras between the two major viral genomes in the original virus isolate, designated common form clone 61E and major variant clone 61C, which were molecularly cloned directly from DNA of the same animal and tissue. Each of three 61E/C chimeras, containing at minimum a 34-amino-acid segment (including a 6-amino-acid insertion and one amino acid substitution) near the C terminus of the 61C surface glycoprotein (gp70), induced fatal immunodeficiency disease in all (12 of 12) infected animals over a course of 33 +/- 10 weeks. By contrast, animals infected with virus 61E, although persistently antigenemic, remained asymptomatic throughout a 48-week observation period. Beginning 14 weeks after infection, a significant decrease (8 to 10%) in the percent of circulating CD4+ T lymphocytes developed in the 61E/C chimera-infected cats, compared with either 61E-infected or control animals. At this time, no significant changes were seen in CD8 cells, B cells, or mitogen-induced blastogenesis. Prior to this initial decline in CD4 cells, the ability of all antigenemic 61E/C-infected cats to generate a primary antibody response to the T-cell-dependent antigen keyhole limpet hemocyanin was markedly impaired, whereas all 61E-infected cats, one 61E/C-infected but nonviremic cat, and all uninfected control cats produced normal antibody responses. The results reported here demonstrate that a major determinant of in vivo immunodeficiency induction by FeLV-FAIDS is contained within a 34-amino-acid C-terminal segment of its surface glycoprotein and that this gp70 alteration determines the early and persistent deficits in CD4+ T lymphocytes and T-cell-dependent antibody responses. We hypothesize that these early immunologic alterations could result from early deletion of a CD4+ helper T-cell subset.

Immunologic abnormalities in pathogen-free cats experimentally infected with feline immunodeficiency virus.

Blood mononuclear cells from 47 cats experimentally infected with feline immunodeficiency virus (FIV) were examined by using monoclonal antibodies directed against feline CD4 and CD8 homologs, a pan-T-cell antigen, and cell surface immunoglobulin. Significant inversion of the CD4+/CD8+ T-cell ratio was observed only in cats that were infected for 18 months or more. This inversion was associated with a decrease in the absolute numbers of CD4+ T cells and a concomitant increase in CD8+ cells. However, the total numbers of circulating T and B cells were not significantly reduced. Cats infected with FIV for 24 to 28 months also had significantly elevated levels of serum immunoglobulin G (IgG), but normal levels of IgA and IgM. The long-term decline in CD4+ T cells and hypergammaglobulinemia observed in FIV-infected cats resemble the abnormalities occurring in humans after human immunodeficiency virus infection.

Identification of a CD4 homologue in the cat.

Monoclonal antibody, Fel 7, produced against cat T cells, was found to react with a single-chain glycoprotein of Mr 65,000 present on a majority of the thymocytes, 40% of lymph node cells, 15% of splenocytes and 25% of blood mononuclear cells. Using a previously reported antibody that recognizes the feline CD8 antigen, approximately 65% of cat thymocytes were shown to express both the Fel 7 and fCD8 antigens, while 14% and 6% expressed either the Fel 7 or the fCD8 determinant respectively. The Fel 7 and fCD8 antigens were expressed by mutually-exclusive subpopulations of peripheral T cells, and not by B cells, macrophages or other types of blood cells. Expression of the Fel 7 antigen was down-regulated and the molecule was phosphorylated when T cells were stimulated with phorbol ester, while the expression of the fCD8 antigen was unaffected by this treatment. The addition of soluble Fel 7 antibodies efficiently blocked Con A-induced proliferation of T cells in a dose-dependent manner. The data suggest that the mAb Fel 7 identifies a feline CD4 homologue, providing an important reagent for the study of normal and abnormal T cell development in cats.

Feline leukemia virus infection as a potentiating cofactor for the primary and secondary stages of experimentally induced feline immunodeficiency virus infection.

Preexistent feline leukemia virus (FeLV) infection greatly potentiated the severity of the transient primary and chronic secondary stages of feline immunodeficiency virus (FIV) infection. Of 10 FeLV-FIV carrier cats, 5 died of experimentally induced FIV infection, compared with 2 deaths in 10 cats infected only with FeLV and 1 death in 7 cats infected only with FIV. FIV-infected cats with preexistent FeLV infections developed severe depression, anorexia, fever, diarrhea, dehydration, weight loss, and leukopenia 4 to 6 weeks after infection and were moribund within 2 weeks of the onset of signs, whereas cats infected only with FIV developed much milder self-limiting gross and hematologic abnormalities. Pathologic findings in dually infected cats that died were similar to those observed previously in cats dying from uncomplicated primary FIV infection but were much more widespread and severe. Coinfection of asymptomatic FeLV carrier cats with FIV did not increase the levels of FeLV p27 antigen present in their blood over that seen in cats infected with FeLV alone. The amount of proviral FIV DNA was much higher, however, in dually infected cats than in cats infected only with FIV; there was a greater expression of FIV DNA in lymphoid tissues, where the genome was normally detected, and in nonlymphoid tissues, where FIV DNA was not usually found. Dually infedted cats that recovered from the primary stage of FIV infection remained more leukopenic than cats infected with FIV or FeLV alone, and their CD4+/CD8+ T-lymphocyte ratios were inverted. One of these cats developed what was considered to be an opportunistic infection. It was concluded, therefore, that a preexistent FeLV infection in some way enhanced the expression and spread of FIV in the body and increased the severity of both the resulting transient primary and chronic secondary stages of FIV infection. This study also demonstrated the usefulness of the FIV model in studying the role of incidental infectious diseases as cofactors for immunodeficiency-causing lentiviruses.

Structural proteins of equine infectious anemia virus.

Equine infectious anemia virus was found to be comprised of fourteen polypeptides of molecular weight ranging from 10,000 to 79,000. Eighty percent of the virion protein was accounted for by five polypeptides, including two non-glycosylated components (p29 and p13) comprising one-half of the virion protein and three glycoproteins (gp77/79, gp64, and gp40).