Expression and Immunostaining Analyses Suggest that Pneumocystis Primary Homothallism Involves Trophic Cells Displaying Both Plus and Minus Pheromone Receptors.

The genus Pneumocystis encompasses fungal species that colonize mammals' lungs with host specificity. Should the host immune system weaken, the fungal species can cause severe pneumonia. The life cycle of these pathogens is poorly known, mainly because an in vitro culture method has not been established. Both asexual and sexual cycles would occur. Trophic cells, the predominant forms during infection, could multiply asexually but also enter into a sexual cycle. Comparative genomics revealed a single mating type locus, including plus and minus genes, suggesting that primary homothallism involving self-fertility of each strain is the mode of reproduction of Pneumocystis species. We identified and analyzed the expression of the mam2 and map3 genes encoding the receptors for plus and minus pheromones using reverse transcriptase PCR, in both infected mice and bronchoalveolar lavage fluid samples from patients with Pneumocystis pneumonia. Both receptors were most often concomitantly expressed during infection, revealing that both pheromone-receptor systems are involved in the sexual cycle. The map3 transcripts were subject to alternative splicing. Using immunostaining, we investigated the presence of the pheromone receptors at the surfaces of Pneumocystis cells from a patient. The staining tools were first assessed in Saccharomyces cerevisiae displaying the Pneumocystis receptors at their cellular surface. Both receptors were present at the surfaces of the vast majority of the cells that were likely trophic forms. The receptors might have a role in mate recognition and/or postfertilization events. Their presence at the cell surface might facilitate outbreeding versus inbreeding of self-fertile strains.IMPORTANCE The fungi belonging to the genus Pneumocystis may cause severe pneumonia in immunocompromised humans, a disease that can be fatal if not treated. This disease is nowadays one of the most frequent invasive fungal infections worldwide. Whole-genome sequencing revealed that the sexuality of these fungi involves a single partner that can self-fertilize. Here, we report that two receptors recognizing specifically excreted pheromones are involved in this self-fertility within infected human lungs. Using fluorescent antibodies binding specifically to these receptors, we observed that most often, the fungal cells display both receptors at their surface. These pheromone-receptor systems might play a role in mate recognition and/or postfertilization events. They constitute an integral part of the Pneumocystis obligate sexuality within human lungs, a cycle that is necessary for the dissemination of the fungus to new individuals.

Site-Directed Mutagenesis of the 1,3-ß-Glucan Synthase Catalytic Subunit of Pneumocystis jirovecii and Susceptibility Assays Suggest Its Sensitivity to Caspofungin.

The echinocandin caspofungin inhibits the catalytic subunit Gsc1 of the enzymatic complex synthesizing 1,3-ß-glucan, an essential compound of the fungal wall. Studies with rodents showed that caspofungin is effective against Pneumocystis asci. However, its efficacy against asci of Pneumocystis jirovecii, the species infecting exclusively humans, remains controversial. The aim of this study was to assess the sensitivity to caspofungin of the P. jirovecii Gsc1 subunit, as well as of those of Pneumocystis carinii and Pneumocystis murina infecting, respectively, rats and mice. In the absence of an established in vitro culture method for Pneumocystis species, we used functional complementation of the Saccharomyces cerevisiae gsc1 deletant. In the fungal pathogen Candida albicans, mutations leading to amino acid substitutions in Gsc1 confer resistance to caspofungin. We introduced the corresponding mutations into the Pneumocystis gsc1 genes using site-directed mutagenesis. In spot dilution tests, the sensitivity to caspofungin of the complemented strains decreased with the number of mutations introduced, suggesting that the wild-type enzymes are sensitive. The MICs of caspofungin determined by Etest and YeastOne for strains complemented with Pneumocystis enzymes (respectively, 0.125 and 0.12 µg/ml) were identical to those upon complementation with the enzyme of C. albicans, for which caspofungin presents low MICs. However, they were lower than the MICs upon complementation with the enzyme of the resistant species Candida parapsilosis (0.19 and 0.25 µg/ml). Sensitivity levels of Gsc1 enzymes of the three Pneumocystis species were similar. Our results suggest that P. jirovecii is sensitive to caspofungin during infections, as are P. carinii and P. murina.

Functional and Expression Analyses of the Pneumocystis MAT Genes Suggest Obligate Sexuality through Primary Homothallism within Host Lungs.

Fungi of the genus Pneumocystis are obligate parasites that colonize mammals' lungs and are host species specific. Pneumocystis jirovecii and Pneumocystis carinii infect, respectively, humans and rats. They can turn into opportunistic pathogens in immunosuppressed hosts, causing severe pneumonia. Their cell cycle is poorly known, mainly because of the absence of an established method of culture in vitro It is thought to include both asexual and sexual phases. Comparative genomic analysis suggested that their mode of sexual reproduction is primary homothallism involving a single mating type (MAT) locus encompassing plus and minus genes (matMc, matMi, and matPi; Almeida et al., mBio 6:e02250-14, 2015). Thus, each strain would be capable of sexual reproduction alone (self-fertility). However, this is a working hypothesis derived from computational analyses that is, in addition, based on the genome sequences of single isolates. Here, we tested this hypothesis in the wet laboratory. The function of the P. jirovecii and P. carinii matMc genes was ascertained by restoration of sporulation in the corresponding mutant of fission yeast. Using PCR, we found the same single MAT locus in all P. jirovecii isolates and showed that all three MAT genes are often concomitantly expressed during pneumonia. Extensive homology searches did not identify other types of MAT transcription factors in the genomes or cis-acting motifs flanking the MAT locus that could have been involved in MAT switching or silencing. Our observations suggest that Pneumocystis sexuality through primary homothallism is obligate within host lungs to complete the cell cycle, i.e., produce asci necessary for airborne transmission to new hosts.IMPORTANCE Fungi of the genus Pneumocystis colonize the lungs of mammals. In immunosuppressed human hosts, Pneumocystis jirovecii may cause severe pneumonia that can be fatal. This disease is one of the most frequent life-threatening invasive fungal infections in humans. The analysis of the genome sequences of these uncultivable pathogens suggested that their sexual reproduction involves a single partner (self-fertilization). Here, we report laboratory experiments that support this hypothesis. The function of the three genes responsible for sexual differentiation was ascertained by the restoration of sexual reproduction in the corresponding mutant of another fungus. As predicted by self-fertilization, all P. jirovecii isolates harbored the same three genes that were often concomitantly expressed within human lungs during infection. Our observations suggest that the sexuality of these pathogens relies on the self-fertility of each isolate and is obligate within host lungs to complete the cell cycle and allow dissemination of the fungus to new hosts.

Functional characterization of the Pneumocystis jirovecii potential drug targets dhfs and abz2 involved in folate biosynthesis.

Pneumocystis species are fungal parasites colonizing mammal lungs with strict host specificity. Pneumocystis jirovecii is the human-specific species and can turn into an opportunistic pathogen causing severe pneumonia in immunocompromised individuals. This disease is currently the second most frequent life-threatening invasive fungal infection worldwide. The most efficient drug, cotrimoxazole, presents serious side effects, and resistance to this drug is emerging. The search for new targets for the development of new drugs is thus of utmost importance. The recent release of the P. jirovecii genome sequence opens a new era for this task. It can now be carried out on the actual targets to be inhibited instead of on those of the relatively distant model Pneumocystis carinii, the species infecting rats. We focused on the folic acid biosynthesis pathway because (i) it is widely used for efficient therapeutic intervention, and (ii) it involves several enzymes that are essential for the pathogen and have no human counterparts. In this study, we report the identification of two such potential targets within the genome of P. jirovecii, the dihydrofolate synthase (dhfs) and the aminodeoxychorismate lyase (abz2). The function of these enzymes was demonstrated by the rescue of the null allele of the orthologous gene of Saccharomyces cerevisiae.

Southern Buenos Aires stroke project.

BACKGROUND: Ethnic differences and vascular risk factors are the major determinants of stroke subtypes. Nevertheless, specific data from undeveloped countries is difficult to obtain. Natives from South America may have a higher frequency of penetrating small vessel disease and hemorrhagic stroke. However, there are few studies in South America supporting these findings. OBJECTIVE: We analyze demographic, ethnic, risk factors, clinical characteristics, and stroke subtypes in all patients with acute stroke admitted to our hospital. METHODS: We studied all consecutive acute stroke patients admitted to the Ramos Mejia Hospital in Buenos Aires from 1997 to 1999. Our hospital serves a determined population of Southern Buenos Aires. Data were collected prospectively on patients' admission in a form especially designed for this study including vascular risk factors, clinical features, epidemiological characteristics, and neuroradiological findings. Stroke subtypes were determined according to the TOAST classification. RESULTS: Among 361 acute stroke patients, 31% had hemorrhagic stroke. It was more frequent among Natives (34%) than Caucasians (27%) (P<0.002). Ischemic stroke subtypes were as follows: 105 (42%) patients had lacunar, 31 (12%) atherosclerotic stroke, 53 (21%) cardioembolic infarction, and 16 (6%) other causes of stroke. Forty-five (18%) patients were classified as undetermined. Small vessel disease was higher among Caucasians (35%) than Natives (24%). CONCLUSIONS: Penetrating artery disease (42%) and intracranial hemorrhage (31%) were the most common stroke subtypes, being more frequent than reported in the literature. Natives had significantly higher frequency of hemorrhagic stroke than Caucasians.

[Myelodysplastic syndromes. Clinico-pathologic analysis of 54 cases]. / Sindromi mielodisplastiche. Analisi clinico-patologica di 54 casi.

For myelodysplastic syndromes (MDS) many scoring systems were developed to improve the prognostic stratification of patients. METHODS AND AIM OF THE STUDY: We enrolled 54 primary MDS patients, having an advanced median age of 78.5 years, that discouraged the choice of aggressive treatments. Then, we employed only the Bournemouth score (without cytogenetic analysis), with the aim to identify the best predictor of death and of AML development. RESULTS: Both for overall and leukemia-free analysis, shorter survival and faster development of AML were found in MDS patients with severe peripheral cytopenia, RAEB-T, major proportion of bone marrow blasts (over 20%) and with the higher Bournemouth score (i.e. 3-4). The multivariate analysis by the Cox's regression model showed that a high percentage of bone marrow blasts presented the best statistical significance. CONCLUSIONS: In our survey we confirmed the value of Bournemouth score in identifying those MDS patients presenting a greater risk of death and AML development. Moreover, a high percentage of bone marrow blasts rose as the best predictor of death and leukemic evolution. The kariotypic analysis, with a stronger prognostic power but also complex and expensive, was not performed in our elderly MDS patients, who were unable to tolerate aggressive treatments.

DCEP (dexamethasone, cyclophosphamide, etoposide, and cisplatin) is an effective regimen for peripheral blood stem cell collection in multiple myeloma.

DCEP (dexamethasone, cyclophosphamide, etoposide, and cisplatin) has proved to be an effective salvage therapy for refractory-relapsed MM patients. Little is known, however, about its potential as mobilizing therapy. The aim of this study was to evaluate the efficacy of DCEP in mobilizing PBSC and to define its toxicity. Fifty-five MM patients received DCEP followed by G-CSF as part of high-dose programs including autologous transplantation. At the time of mobilization, 40 patients had previously received VAD only, and 15 alkylating agents. Mobilization was successful (minimum number of CD34(+) cells 2 x 10(6)/kg) in 48/55 patients (87%), and 41/55 patients (75%) collected >4 x 10(6)/kg CD34(+) cells. Of the seven patients who did not mobilize stem cells, five (71%) had been previously exposed to alkylating agents. The median number of CD34(+) cells harvested was 5.8 x 10(6)/kg (range 2.1-22.4). There was no treatment-related mortality. The side-effects of DCEP were always tolerable. No neutropenia <1000/microl nor thrombocytopenia <50,000/microl were observed. No patient required transfusion as a consequence of therapy, or hospitalization for septic complications. In conclusion, DCEP, in addition to its demonstrated anti-tumor activity, is an effective regimen for mobilizing peripheral blood progenitor cells in myeloma patients, with little or no side-effects. These properties render DCEP a useful regimen for the debulking and mobilization phase of high-dose programs for multiple myeloma.

Differences in stroke subtypes among natives and caucasians in Boston and Buenos Aires.

BACKGROUND AND PURPOSE: Several issues regarding ethnic-cultural factors, sex-related variation, and risk factors for stroke have been described in the literature. However, there have been no prospective studies comparing ethnic differences and stroke subtypes between populations from South America and North America. It has been suggested that natives from Buenos Aires, Argentina, may have higher frequency of hemorrhagic strokes and penetrating artery disease than North American subjects. The aim of this study was to validate this hypothesis. METHODS: We studied the database of all consecutive acute stroke patients admitted to the Ramos Mejia Hospital (RMH) in Buenos Aires and to the Beth Israel Deaconess Medical Center (BIMC) in Boston, Massachusetts, from July 1997 to March 1999. Stroke subtypes were classified according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria. All information on patients (demographic, clinical, and radiographic) was recorded prospective to the assessment of the stroke subtype. RESULTS: Three hundred sixty-one and 479 stroke patients were included at RMH and BIMC stroke data banks, respectively. Coronary artery disease was significantly more frequent in BIMC (P:<0.001), whereas tobacco and alcohol intake were significantly more frequent in RMH (P:<0.001). Intracerebral hemorrhage (P:<0.001) and penetrating artery disease (P:<0.001) were significantly more frequent in the RMH registry, whereas large-artery disease (P:<0.02) and cardioembolism (P:<0.001) were more common in the BIMC data bank. CONCLUSIONS: Penetrating artery disease and intracerebral hemorrhage were the most frequent stroke subtypes in natives from Buenos Aires. Lacunar strokes and intracerebral hemorrhage were more frequent among Caucasians from Buenos Aires than Caucasians from Boston. Poor risk factor control and dietary habits could explain these differences.

[Primary extranodal non-Hodgkin's lymphoma of muscle tissue]. / Linfoma non-Hodgkin extranodale primitivo del tessuto muscolare.

Primary muscular involvement is extremely rare in non-Hodgkin's lymphomas. To our knowledge few cases are reported in literature and all of them concern patients with unifocal muscular lymphoid masses usually growing in one of the extremities. Our case-report, instead, regards a 78 years-old woman presenting primary multifocal muscular involvement by extranodal non-Hodgkin's lymphoma (right upper and lower limbs affected at the same time). Therefore, in contrast with the therapeutic approach suggested by other Authors in such neoplasms (radiotherapy or combined radio-chemotherapy), we preferred to administer only chemotherapy. The treatment led to a complete regression of all lymphoid masses. By now the woman is healthy and disease-free as confirmed at the one-year haematological follow-up.

Combined differentiating therapy for myelodysplastic syndromes: a phase II study.

An in vitro synergism between different inducers of AML cell differentiation has been previously observed. Therefore, we treated 53 myelodysplastic (MDS) patients with a low dose combination of cis-retinoic acid (cRA, 20-40 mg/day) and 1,25 alpha (OH)2 cholecalciferol [(OH)2D3, 1-1.5 micrograms/day] +/- intermittent 6-thioguanine (30 mg/m2/day). The latter was reserved for patients with bone marrow (BM) blast excess (> or = 5%). The treatment was well tolerated, without major toxicity. Among 25 patients with BM blasts less than 5%, we observed one complete, eight partial and four minor responses (response rate 52%) with a median response duration of 8 months (2 +/- 24). Median survival, which did not correlate with response, is projected at 76 months. Thirty-one patients with BM blast excess (> or = 5%), including three of the previous group who progressed to refractory anemia with excess of blasts (RAEB), were treated with the three-drug protocol. One complete, 12 partial and six minor responses were obtained (response rate 61%) with a median response duration of 6 months (2-29+). A significant difference in survival (P < 0.005) was observed between the 19 responders (median 25 months) and the 12 non-responders (median 9 months). A reduction in the transfusion need was observed in 41% of the transfusion-dependent patients with blast excess and in 53% of those without blast excess. Therefore, combined differentiating therapy seems more effective than previously reported single agent treatments and should be considered for a larger randomized study to assess its actual impact on survival of MDS patients.

[Occurrence of primary hypothyroidism in alpha-interferon treatment]. / Insorgenza di ipotiroidismo primitivo in corso di terapia con alfa-interferone.

In recent years recombinant alpha interferon (IFN) has been widely used in the treatment of neoplastic and infectious diseases. Induced autoimmune disorders and thyroid impairment are getting increasing relevance in the field of side-effects complicating long-term alpha-interferon courses. We monitored thyroid function in 35 patients receiving alpha-IFN therapy for different diseases (chronic hepatitis, essential thrombocytemia, multiple myeloma, chronic myeloid leukemia, essential polycytemia, essential crioglobulinemia and hairy-cell leukemia). All of them were euthyroid and negative for anti-thyroid serum antibodies before treatment. Six months later, 6 patients (17%) developed primary hypothyroidism with elevated antithyroid antibodies in 5 cases; 1 continuing to be negative. None of our patients developed hyperthyroidism. Overall, "indirect-autoimmune" and "direct non autoimmune" mechanisms are considered possible and/or combined pathogenetic moments of thyroid disfunction during alpha-IFN therapy. Thyroid complications mainly occur when latent impairment of immune system exists. Thyroid circulating hormones levels and autoimmunity screening should be performed in all patients before starting and during long-term alpha-IFN treatment.

[The neuroglycopenic syndrome due to insulinoma. A clinical case report]. / Sindrome neuroglicopenica da insulinoma. Descrizione di un caso clinico.

The authors report a case of a woman affected by pancreatic insulinoma who had been suffering from recurrent and misdiagnosed hypoglycemic attacks since 3 years. The total loss of warning neurogenic symptoms replaced by sudden onset of neuroglycopenic symptoms had delayed the proper and early diagnosis because of repeated and useless cardiovascular and neurological investigations. Moreover, it is stressed how difficult is to reveal such neoplasia that, despite the severe symptoms, are usually small in size and often undetectable even with TC scan. Therefore, when clinical pattern is strongly suggestive for insulinoma the use of invasive angiography or other techniques is mandatory and often conclusive. Actually clinical data have the priority in whole diagnostic pathway.

[Immunological indices in myelodysplastic syndromes]. / Indici immunologici nelle sindromi mielodisplastiche.

Several studies aiming to describe the immunological abnormalities occurring in patients affected by myelodysplastic syndromes (MDS) have been carried out in recent years. We report on the immunological abnormalities found in 22 myelodysplastic patients at the time of diagnosis (RA: 2 cases; ASIA: 4 cases; AREB: 6 cases; AREB-T: 6 cases. LMMC: 4 cases). Matched with similarly aged healthy people (controls) all our patients revealed a significant lymphocytopenia mainly due to a reduction both in number and percentage of T-helper series with decreased OK T4/OK T8 ratio as a result; even B-cells were reduced in number but their percentage still overlapped with the controls. Out of 22 patients, 13 showed hypergammaglobulinemia (polyclonal in 12 cases, monoclonal in the one left) and 2 read positive for Coomb's and Ana-test respectively. The involvement of T-cell immunity in the course of MDS can be explained if we consider the clonal origin of such diseases. Among myelodysplastic patients the ones affected by LMMC, AREB and AREB-T show the heaviest immunological abnormalities: in these cases the whole of T-cells subsets and NK cells as well are affected. Eventually, the mentioned abnormalities are of paramount importance to explain how easily these patients can develop both severe infectious diseases and abrupt acute leukemia.

[Partial remission of non-Hodgkin's lymphoma (stage IV-B) following immunostimulating therapy with thymopentin]. / Remissione parziale di LMNH stadio IV-B in seguito a terapia immunostimolante con timopentina.

Therapy of low-grade of malignancy non-Hodgkin's lymphoma in an advanced stage is still under discussion: aggressive poly-chemotherapies, such as radiotherapy and conventional chemotherapies did not prove to be more effective than conservative treatments. We report the case of a woman suffering from a low-grade of malignancy non-Hodgkin's lymphoma (stage IV-B). She was in such bad general conditions that she could not be treated with chemotherapy. She received an immunostimulating drug, thymopentin for 10 months. After this treatment, the general condition of the patient was improved and a partial remission of the lymphoproliferative disease was observed. The patient is still in constant fairly good health.

[Determination of lymphocyte subpopulations, defined with monoclonal antibodies, in patients with iron deficiency anemia]. / Determinazione delle sottopopolazioni linfocitarie, definite con anticorpi monoclonali, in pazienti con anemia sideropenica.

Lymphocytary subpopulations have been determined, by means of monoclonal antibodies, in 24 patients afflicted with iron deficient anaemia, repeating the determination in 15 of the patients after an adequate iron therapy. In iron deficient patients a significant reduction of the percentage of lymphocytes T (OKT3+) has been observed, in comparison with normal controls, as well as a significant reduction of the percentage of cells OKT8+, with a significant increase of the OKT4+/OKT8+ ratio. In the 10 patients who at the moment of the revaluation did not present any longer either anaemia or iron deficiency, the percentage of lymphocytes OKT3+ and OKT8+ had returned to normal values, as well as the OKT4+/OKT8+ ratio. The 5 patients still resulting anaemic in course of control presented, on the contrary, a significant reduction of cells OKT3+ and OKT8+, while the OKT4+/OKT8+ ratio resulted lightly increased, however not significantly.

[Determination of lymphocyte subpopulations in multiple myeloma and monoclonal gammopathies of undetermined significance]. / Determinazione delle sottopopolazioni linfocitarie nel mieloma multiplo e nelle gammopatie monoclonali di significato indeterminato.

By means of monoclonal antibodies, the lymphocyte subpopulation in 12 patients affected by multiple myeloma, 3 by Waldenström's macroglobulinemia, 7 by M.G.U.S. and 8 patients affected by accompanying monoclonal gammopathy have been determined. The group of patients affected by multiple myeloma or Waldenström's macroglobulinemia presented, against control, a significant reduction in the OK T3+ and OK T4+ cell percentage, with a remarkable reduction of the OK T4+/OK T8+ ratio. The OK T8+ cell average, if considered as an absolute value, was not modified in comparison to normal value, while the absolute number of OK T4+ cells was substantially reduced. No significant modifications have been ascertained in the percentage of SIg+ cells. The patients affected by M.G.U.S. did not present significant difference against controls, with regard to the lymphocyte number and the percentage of OK T3+ and OK T4+ cells. On the contrary, a significant increase of the percentage and absolute number of OK T8+ cells was observed. Also in these cases, a significant reduction of the OK T4+/OK T8+ ratio was observed. Finally, the patients affected by accompanying monoclonal gammopathy presented a significant reduction of OK T3+ and OK T8+ cells, with an increase of the percentage of OK T4+ cells and of the OK T4+/OK T8+ ratio.

[Systemic mastocytosis without skin involvement. Presentation of a case and review of the literature]. / Mastocitosi sistemica senza interessamento cutaneo. Presentazione di un caso e revisione della letteratura.

A case of SMCD without apparent cutaneous involvement is described. It has however been possible to demonstrate, by a test of cutaneous biopsy at the optical and electron microscope, an increased number of aberrant mast cells, together with a cutaneous melanosis due to the intense melanin deposit. The meaning of this association is unknown and never previously notified in the literature.

[Behavior of PGE1 and PGE2 in the granulocytes of normal and leukemic subjects during phagocytosis in vitro]. / Comportamento della PGE1 e della PGE2 nei granulociti di soggetti normali e leucemici durante la fagocitosi in vitro.

The behaviour of phagocytosis and that of PGE1 and PGE2 in the circulating granulocytes of normal and leukaemic subjects was investigated by the comparison of latex particles and the PAP (peroxidase-antiperoxidase) immuno-enzymatic method respectively. Generally speaking, it was found that chronic myeloid leukaemia and acute myeloblastic leukaemia were accompanied by a marked reduction in phagocyting capacity, whereas this is apparently normal in CLL and ALL. PCE values, on the other hand, were well down in lymphatic leukaemia, AML and AMML, but not in CML, where high PGE (especially PGE2) was noted both basally and after phagocytosis. That the PGE take part in phagocytosis is shown by their redistribution in phagocyting cells, with elective accumulation in the membrane and around the engulfed material.