[A prospective multi-center clinical investigation of HIV-negative pulmonary cryptococcosis in China].

Objective: To investigate the current status of the diagnosis and treatment of pulmonary cryptococcosis in respiratory medicine and improve the understanding of the clinical characteristics of HIV-negative pulmonary cryptococcosis in China. Methods: A prospective multi-center open cohort study was designed to screen for pulmonary cryptococcosis in the general wards and intensive care units of the Department of Respiratory Diseases in 22 hospitals. The HIV-negative patients with positive cryptococcal etiological diagnosis based on smear culture, antigen detection and histopathology were enrolled in the study. The clinical data of enrolled patients were collected and analyzed. Results: A total of 457 cases of pulmonary cryptococcosis were enrolled, among which 3.28% (15/457) were disseminated infections. The case fatality rate was 0.88% (4/457). The majority of the cases were diagnosed by histopathological examinations (74.40%, 340/457) and cryptococcus antigen detection (37.64%, 172/457). Patients with pulmonary cryptococcosis accounted for 2.04‰ (457/223 748) of the total hospitalized patients in the Department of Respiratory Diseases during the same period, and the ratio was the highest in south and east China. Meanwhile, 70.24% (321/457) of the patients had no underlying diseases, while 87.75% (401/457) were found to have immunocompetent status. Cough and expectoration were the most common clinical symptoms in patients with pulmonary cryptococcosis. However, 25.16% (115/457) of the patients had no clinical symptom or physical signs. In terms of imaging features on pulmonary CT, multiple pulmonary lesions were more common than isolated lesions, and there were more subpleural lesions than perihilar or medial lesions. Morphologically, most of the lesions were middle-sized nodules (1-5 cm) or small-sized nodules (3 mm to 1 cm). The sensitivity of serum cryptococcus antigen test was 71.99% (203/282). Moreover, antigen-positive patients differed from antigen-negative patients in terms of basic immune status, clinical symptoms, imaging features and infection types. Meanwhile, immunocompromised patients differed from immunocompetent patients in terms of clinical symptoms, physical signs, infection-related inflammation indicator levels, imaging features, serum cryptococcus antigen positive rate and prognosis. Conclusions: The majority of cases of HIV-negative pulmonary cryptococcosis in China had no underlying disease or immunocompromised status, and the overrall prognosis was favorable. However, early diagnosis of HIV-negative pulmonary cryptococcosis remains challenging due to the complicated manifestations of the disease.

CD4+Foxp3+ regulatory T-cell impairment by paclitaxel is independent of toll-like receptor 4.

Paclitaxel (PTX) is one of the most widely used clinical antitumour drugs in chemotherapy nowadays. Its effect on immune system has become a hot spot of research in recent years. Here, we demonstrated that PTX not only decreased the percentage of CD4+Foxp3+ regulatory T (Treg) cells both in vitro and in vivo but also impaired cell viability and cytokine production of Treg cells rather than CD4+Foxp3- effector T (Teff) cells. As PTX has been reported to mimic the activity of LPS to trigger the toll-like receptor 4 (TLR4) signalling pathway in macrophages, we investigated the possible role of TLR4 in the effect of PTX. However, although TLR4 expression on Treg cells was higher than that on Teff cells, the expression level remained unaltered in both Treg and Teff cells after PTX treatment. Surface molecules and activation markers in Treg and Teff cells did not change, either. Further study showed that the effect of PTX on TLR4-/- mice deficient in TLR4 signalling was similar to that on C57BL/6 mice both in vivo and in vitro. These data indicate that the selective impairment of Treg cells by PTX is independent of TLR4.

DNA hypomethylation is crucial for apoptotic DNA to induce systemic lupus erythematosus-like autoimmune disease in SLE-non-susceptible mice.

OBJECTIVES: Systemic lupus erythematosus (SLE) is characterized by serological presence of anti-double-stranded DNA (dsDNA) antibodies and its pathogenesis remains unclarified. Our previous work found that syngeneic activated lymphocyte-derived DNA (ALD-DNA) induced SLE-like autoimmune disease in the SLE-non-prone BALB/c mice. Here, the biological and chemical characteristics of the somatic DNA were focused upon to investigate their contribution to the autoimmunity induction to provide clues for the understanding of the pathogenesis of SLE in non-susceptible strains. METHODS: Induction of anti-dsDNA antibodies, glomerulonephritis and proteinuria was evaluated in BALB/c mice after subcutaneous immunization with apoptotic DNA (annexin-V+) extracted from concanavalin A or UV-treated apoptotic splenocytes or necrotic DNA from necrotic splenocytes. The hypomethylated apoptotic DNA and the normal DNA were then methylated and demethylated, respectively, by CpG methylase or 5-azacytidine treatment to re-evaluate their immunogenicity in BALB/c mice. RESULTS: It was apoptotic but not necrotic DNA that induced SLE-like autoimmune disease and the level of apoptotic DNA was associated with the level of anti-dsDNA antibodies. The apoptotic DNA exhibited significantly lower methylation levels than the normal DNA. Methylation of the hypomethylated apoptotic DNA significantly impaired its ability to induce anti-dsDNA antibodies and proteinuria, while demethylation of the normal or necrotic DNA endowed them with the immunogenicity to induce the SLE-like syndrome. CONCLUSIONS: Our study provides direct evidence showing that DNA hypomethylation is essential for apoptotic DNA to induce SLE-like autoimmune disease in non-susceptible mice, which may help in elucidating the pathogenesis of SLE.

[Research progress in gene engineered antibodies].

Gene engineered antibodies(GEAs) have been applied in a lot of aspects with the great development of the technique of GEAs. The technique of GEAs such as ribosome display and phage surface display and their application progress were summarized in this review.

Induction of systemic lupus erythematosus-like syndrome in syngeneic mice by immunization with activated lymphocyte-derived DNA.

OBJECTIVES: Systemic lupus erythematosus (SLE) is the prototype of autoimmune disease and the mechanisms underlying the disease have not yet been elucidated. Thus, animal models of SLE would facilitate investigation of pathogenetic mechanisms involved in the development of the disease. This study characterizes a murine model of SLE-like syndrome induced by syngeneic activated lymphocyte-derived DNA (referred to as ALD DNA). METHODS: Normal BALB/c mice were immunized subcutaneously with highly purified ALD DNA. Anti-double-stranded DNA (anti-dsDNA) antibodies were determined by enzyme-linked immunosorbent assay. Other SLE-associated autoantibodies were examined by indirect immunofluorescence and anti-ENA (extractable nuclear antigen) profile assay. Pathological changes were analysed by light microscopy and electron microscopy. Kidney cryostat sections were viewed by immunofluorescence for the presence of glomerular IgG and C3 deposits. Proteinuria was measured by Coomassie brilliant blue assay. RESULTS: High levels of anti-dsDNA antibodies and other autoantibodies frequently appearing in SLE were detectable in the sera of ALD DNA-immunized mice. Glomerulonephritis and glomerular deposition of IgG plus C3 were observed in the kidney sections. Moreover, proteinuria was seen in the immunized mice. CONCLUSIONS: SLE-like syndrome can be induced by ALD DNA in normal mice. This induced model may be useful for elucidating the mechanisms involved in autoimmunity to DNA and the development of SLE.

Solid matrix-antibody-antigen complex can clear viraemia and antigenaemia in persistent duck hepatitis B virus infection.

One-day-old ducklings experimentally infected with duck hepatitis B virus (DHBV) were found to be immunologically tolerant to virus antigens (DHBsAg, DHBcAg), with no humoral or cellular immune responses being detected. When immunized with virus antigens in Freund's complete adjuvant, no immune responses could be induced. Rabbit anti-DHBs sera were complexed to a solid matrix (Staphylococcus aureus Cowan A strain) and purified DHBsAg was bound to this complex to form a solid matrix antibody-antigen (SMAA) complex. This SMAA was used as an immunogen to immunize the tolerant ducks. After three injections, in 12 of 17 ducks serum DHBV DNA became absent and serum DHBsAg was cleared. In eight of 16 ducks, low titres of anti-DHBs could be detected. The SMAA approach shows potential for application in immunoregulatory treatment for chronically infected hepatitis B patients.

Frequency of hepatitis B virus e-minus mutants varies among patients from different areas of China.

Four hundred forty-six serum samples from HBsAg-positive chronic hepatitis B patients were collected from five areas in China (eastern coastal city, Shanghai; southwestern inland city, Chengdu; mid-inland city, Wuhan; southern island city, Haikou; and northeastern city, Changchun). Precore stop codon variants (e-minus mutants) were screened using a rapid method of polymerase chain reaction (PCR) amplification of a precore and partial core gene fragment (nucleotides 1785-2172), followed by dot-blot hybridization with specific oligonucleotide probes (M0, and M1 + M2). The sequence of the M0 probe covered the distal precore region of wild-type virus (nucleotides 1887-1908), and the sequences of the M1 and M2 probes were from sequences mutated at nt. 1898, (TGG-->TAG) with or without additional change at nt. 1901. A significantly lower incidence of the precore stop codon was found in anti-HBe-positive serum samples from Haikou (17.6%), whereas in other areas the percentages of this mutation in anti-HBe positive sera ranged from 47.4% to 78.9%. In HBeAg-positive samples, the rate of e-minus mutant in coexistence with wild-type virus was low in specimens from Haikou (9.5%) and Changchun (2.9%) compared to other areas in China. In contrast, coexistence of mutant and wild-type virus was frequently detected in samples from Wuhan (50.0%).