EXPERIMENTAL PARACOCCIDIOIDOMYCOSIS IN PREGNANT RATS

Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioidesbrasiliensis (Pb), is the most prevalent systemic mycosis in Latin America. There arefew reports in the literature about the disease damages during pregnancy and theconsequences to the fetuses and breeding. This study evaluated the implications ofPCM during pregnancy on offspring and mothers in Wistar rats. Groups of rats weresubmitted to systemic Pb infection, by intraperitoneal infusion, and mated 30 daysafter the infection date. Immediately after birth, rats and neonates were sacrificedto obtain organs for standard histological examination, morphometric analysis, fungirecovery by plating (CFU) and dosing of anti-Pb antibodies by ELISA. There were nostillbirths or miscarriages, however, the fetuses from infected pregnant rats hadlower body and organ weight but the fertility rate was 100%. The largest number ofCFU was recovered from the organ of pregnant rats, the pathological examinationrevealed more severe infection in the same group, further on the largest number ofgranulomas and fungal field. It can be concluded that the PCM was more severe in thegroup of pregnant rats, with implications to the weight of offspring.     

Neuroparacoccidioidomycosis: analysis of 13 cases observed in an endemic area in Brazil

The epidemiological, clinical and laboratory features of 13 cases of neuroparacoccidioidomycosis (NPCM) were analysed. All patients were men, with a mean age of 41.6 years. The lungs were involved in 11 cases (84.6%) and only two cases had mycosis limited to the central nervous system. Co-morbidity was observed in four patients (malignant neoplasm in three and diabetes mellitus in one). The most frequent neurological manifestations were paresis (eight cases), headache (five cases) and gait disturbance (four cases). Neuroimaging diagnosis showed a predominance of multiple round lesions with ring enhancement following contrast medium injection. Lesions were seen in the brain hemispheres (nine cases), thalamus (nine cases), cerebellum (four cases), brainstem (four cases) and spinal cord (four cases). Most cases responded well to therapy. Lesions with enhancement following contrast medium injection persisted in four patients for a period of 6 months to 8 years. These findings emphasize the importance of considering NPCM in the differential diagnosis of brain and spinal cord lesions in endemic areas of paracoccidioidomycosis.     

国内首例慢性播散性副球孢子菌病

报道国内首例慢性播散性副球孢子菌病。患者男, 49岁, 因皮肤丘疹、结节1年, 口腔黏膜丘疹、溃疡2个月入院。皮肤科检查:左足水肿, 左足底多发溃疡, 表面结痂, 左足第3、4趾间及第4、5趾间溃疡, 基底呈颗粒状, 伴点状出血、渗出;左足背、左足内侧及左膝多发丘疹、结节、斑块, 中央溃疡、结痂;左腕部2个丘疹, 上唇左侧1个丘疹, 表面结痂;牙龈、颊黏膜、唇黏膜及上颚可见红色斑块伴溃疡、点状出血, 皮损以左侧为主。浅表淋巴结彩超:双侧颈部及锁骨上窝淋巴结肿大, 左侧为著。胸腹部计算机断层扫描图像示:双肺弥漫粟粒样结节影及条索状、云絮状、结节状高密度影, 左侧肾上腺明显增粗。口腔、左下肢皮损组织真菌免疫荧光染色, 可见酵母细胞。口腔黏膜、左下肢皮损组织病理:肉芽肿性炎, 多核巨细胞内外可见酵母细胞, 折射双膜, 无芽、单芽或多芽;过碘酸希夫染色、六胺银染色阳性。左下肢皮损组织真菌培养:25 ℃、37 ℃沙氏葡萄糖琼脂培养基中培养阳性, 均为菌丝相。口腔黏膜及肺泡灌洗液宏基因组学测序:巴西副球孢子菌。诊断:慢性播散性副球孢子菌病。予伊曲康唑胶囊400 mg/d口服, 1个月后皮肤、黏膜皮...     

The role of somatic structure of the fungus Paracoccidioides brasiliensis upon B cell activation in experimental paracoccidioidomycosis.

In this study, we report an increase of the number of antibody-secreting cells and the augmentation of antibody production against unrelated antigens in mice infected with the fungus P. brasiliensis, as well as in mice inoculated with cell wall preparation isolated from P. brasiliensis (CW). The immunomodulatory effect of the live fungus and the CW preparation was dose-dependent, and their actions were mainly restricted to the i.v. or i.p. inoculation simultaneously with the sheep erythrocyte challenge by the i.v. route or restricted to i.p. inoculation of CW when bovine serum albumin (BSA) antigen was used. The dependence of antibody production on different routes of CW inoculation was correlated with the number of antigen-specific B cells in the spleen as determined by direct and reverse plaque-forming cell assays. The immunization schedules using CW preparation caused a preferential production of IgM and IgG3 antibodies. The results also showed that the hyperactive humoral immune response of mice induced by i.p. inoculation of CW was devoid of polyclonal B cell activation compared with the effects observed for the lipopolysaccharide (LPS)-treated groups. Paracoccidioides brasiliensis CW components may have potent immunological properties related to the non-specific B cell activation found in paracoccidioidomycosis.     

Diagnosis of paracoccidioidomycosis by a dot blot assay using a recombinant Paracoccidioides brasiliensis p27 protein

A variety of immunological methods have proven useful for Paracoccidioidomycosis (PCM) diagnosis; however, they are often time consuming and many lack sensitivity and specificity, partially attributed to the use of crude antigens, which give cross reactivity. Until now, attempts to clone and express Paracoccidioides brasiliensis immunodominant antigens have presented difficulties of process and problems of cost. In an attempt to obtain a more rapid, sensitive, and specific test for PCM diagnosis, we subcloned the P. brasiliensis p27 gene and used the recombinant protein as the antigen in dot blot assays to evaluate its usefulness in paracoccidioidomicosis diagnosis. The development of an optimised procedure for p27 recombinant protein purification and production led to an easier and less expensive process than the one previously used in our laboratory and allowed the availability of enough purified protein for its evaluation as the antigen in the dot blot assays. In these assays, antibodies present in ten serum samples from seven patients with PCM recognised the recombinant protein showing a sensitivity of 100% with a specificity of 98%. These results confirm the value of the 27-kDa recombinant antigen in the serodiagnosis of paracoccidioidomycosis and that the dot blot format is an alternative to the immunoenzymatic assay procedure.     

The role of HLA antigens in the development of paracoccidioidomycosis

BACKGROUND: Paracoccidioidomycosis is a systemic granulomatous disease that involves primarily the lungs and may disseminate to other organs and systems. It is caused by Paracoccidioides brasiliensis, a fungus that exhibits reversible thermal dimorphism and whose natural habitat is presently unknown. There are two main clinical forms: the acute (subacute) juvenile form and the chronic adult form. The former runs a more rapid course and is more severe than the latter. This mycosis is found throughout Latin America. Brazil accounts for 80% of reported cases. Presumably P. brasiliensis thrives in humid and hot places, especially near forests or farms. The infection is endemic in certain areas, especially in Brazil, Colombia and Venezuela, where nearly 100% of the population show cutaneous paracoccidioidina positive skin tests, indicating previous contact with the fungus, although a small percentage show clinical manifestations of the disease. METHODS: We compared the expression of HLA class I antigens in a healthy group (control) and in a group of patients with paracoccidioidomycosis (chronic adult form) using the Terasaki lymphocytotoxicity test modified by Amos for HLA antigen analysis. AIMS: To discover indications of whether or not individual susceptibility to P. brasiliensis might depend on some specific immunological defect. RESULTS: There is no evidence of association between a specific HLA antigen and paracoccidioidomycosis in the subjects studied. Further investigations are recommended.     

Investigation of the role of complement and complement receptors in the modulation of B cell activation by a Paracoccidioides brasiliensis cell wall fraction

F1 fraction from Paracoccidioides brasiliensis is a potent activator of the complement system. Considering that complement receptors CR1 and CR2 are involved in the regulation of B cell response, we evaluated the in vitro effect of the F1 in the activation of B lymphocytes, as well as the participation of complement receptors in this process. Murine splenocytes were cultured in order to evaluate the expression of CD40, CD45RB and CD69 on B lymphocyte, and IgG and IgM were quantified in the culture supernatant. F1 participated in the activation of B cells, showing a positive modulation effect on all markers analyzed. An increase in the production of IgG was detected in the supernatants when the opsonized F1 fraction was present. Complement receptor blockade with monoclonal antibodies led to a partial reduction in immunoglobulin secretion, suggesting that these receptors, especially CR2, play a role in modulating the function of B lymphocyte stimulated with the opsonized F1 fraction. These results may contribute for a better understanding of the B cell activation and differentiation processes in response to the F1 fraction from P. brasiliensis.     

B and T cell responses elicited by monoclonal anti-idiotypic antibody (Ab2beta) mimicking gp43 from Paracoccidioides brasiliensis

Paracoccidioidomycosis is a systemic mycosis endemic in Latin America, with a high prevalence in Brazil, Argentina, Colombia and Venezuela. The aetiological agent of disease is the thermal dimorphic fungus, Paracoccidioides brasiliensis. A glycoprotein of 43 kD (gp43) is the major antigen of P. brasiliensis. Antibodies directed to this antigen are detected in the sera of all patients with paracoccidioidomycosis (PCM). Recently, it has been shown that mice immunized with anti-gp43 monoclonal antibodies (MAbs) (Ab1), induce the idiotypic cascade in the gp43 system, which produced both, anti-Id antibodies (Ab2) and anti-anti-Id antibodies (Ab3). To further characterize the idiotypic cascade modulation in mice immunized with anti-gp43 MAb 17c, hybridomas were produced. Ab2 MAbs named 7.B12 inhibited (>95%) the binding of gp43 to MAb 17c (Ab1), suggesting that this anti-Id MAb bind to the idiotope, thus fulfilling the internal image criteria. To elucidate whether Ab2 MAb could act as antigen in serological assays, instead of gp43, sera from PCM patients were tested. Using an ELISA test, it was observed that antibodies from patients and not normal serum bound to Ab2. However, the ELISA test using Ab2 bound to the solid phase made possible to serologically monitor the patients after antifungal therapy, showing an equivalent curve when compared with ELISA test employing purified gp43. Our results also showed that, when mice were immunized with Ab2beta and their cells were exposed to gp43 in vitro, a T cell proliferation response was observed.