Identification of Mycobacterium tuberculosis counterimmune (cim) mutants in immunodeficient mice by differential screening

Tuberculosis (TB) is characterized by lifetime persistence of Mycobacterium tuberculosis. Despite the induction of a vigorous host immune response that curtails disease progression in the majority of cases, the organism is not eliminated. Subsequent immunosuppression can lead to reactivation after a prolonged period of clinical latency. Thus, while it is clear that protective immune mechanisms are engaged during M. tuberculosis infection, it also appears that the pathogen has evolved effective countermechanisms. Genetic studies with animal infection models and with patients have revealed a key role for the cytokine gamma interferon (IFN-gamma) in resistance to TB. IFN-gamma activates a large number of antimicrobial pathways. Three of these IFN-gamma-dependent mechanisms have been implicated in defense against M. tuberculosis: inducible nitric oxide synthase (iNOS), phagosome oxidase (phox), and the phagosome-associated GTPase LRG-47. In order to identify bacterial genes that provide protection against specific host immune pathways, we have developed the strategy of differential signature-tagged transposon mutagenesis. Using this approach we have identified three M. tuberculosis genes that are essential for progressive M. tuberculosis growth and rapid lethality in iNOS-deficient mice but not in IFN-gamma-deficient mice. We propose that these genes are involved in pathways that allow M. tuberculosis to counter IFN-gamma-dependent immune mechanisms other than iNOS.     

Scintigraphic and ultrasonographic appearance in different tumor stages of thyroid carcinoma

RATIONALE AND OBJECTIVES: Scintigraphy is routinely used in evaluating thyroid nodules. Functioning nodules are reported to have a low probability of being malignant. Therefore cancer should appear hypo-functioning or "cold" on scintiscan. The aim of the study was to compare the scintigraphic pattern in different tumor stages of thyroid carcinoma. In addition, sonographic results are evaluated. In 151 patients with thyroid carcinoma 99mTc-pertechnetate scans were evaluated retrospectively by a visual inspection scoring method (A = no significant uptake to D = nodular uptake superior to normal thyroid tissue). Planar images were taken using a small field thyroid gamma camera. There were 52 patients with pT1 carcinoma (2 x follicular and 50 x papillary). The mean tumor size was 0.56 +/- 0.26 cm. The scintigraphic results were A and B in 5.7% (n = 6), C in 73% (n = 38), D in 15.6% (n = 8). Out of 40 patients with pT2 carcinoma, 34 had a papillary, 6 a follicular histology. Mean tumor size was 1.66 +/- 0.49 cm. The scintiscan was A in 12.5% (n = 5), B in 32.5% (n = 13), C in 42.5% (n = 17) and D in 12.5% (n = 5). There were 11 patients with pT3 carcinoma (4 x papillary, 7 x follicular). The mean tumor size was 3.96 +/- 0.88 cm in diameter. Scintiscan was A in 72.7% (n = 8), C in 27.3% (n = 3). Among 48 patients with pT4 carcinoma (2 x follicular, 1 x nondifferentiated, 45 x papillary), scan was A in 41.6% (n = 20), B in 14.5% (n = 7), C in 33.3% (n = 16) and D in 10.4% (n = 5). Mean tumor size was 2.16 +/- 1.45 cm (7 carcinomas < or = 1 cm, 23 x 1-2 cm, the remaining > 2 cm). Tumor size plays an important role in routinely used planar scintigraphy. Nodules greater than 2 cm in diameter tend to appear cold but microcarcinomas (< or = 1 cm) are often indifferent on scan. Therefore, planar 99mTc-pertechnetate scintigraphy is of little value in evaluating small thyroid nodules. In order to diagnose small thyroid nodules, ultrasonography and ultrasonographically guided FNAB should be recommended as the initial diagnostic steps in clinical routine.     

Biochemical and immunological characterization of MP65, a major mannoprotein antigen of the opportunistic human pathogen Candida albicans

In the search of the antigenic determinants of a 65-kDa mannoprotein (MP65) of Candida albicans, tryptic fragments of immunoaffinity-purified MP65 preparations were tested for their ability to induce lymphoproliferation of human peripheral blood mononuclear cells (PBMC). Five major peptides (T1 to T5) were shown to induce a vigorous proliferation of PBMC from the majority of the eight healthy human subjects tested. With the use of synthetic peptides, critical amino acid sequences of the two most immunoactive (T1 and T2) peptides were determined. Similar to what was found for the MP65 molecule, no PBMC multiplication was induced by the antigenic peptides in cultures of naive cord blood cells. The amino acid sequence analysis of tryptic and chymotryptic peptides of MP65 demonstrated a substantial homology with the deduced sequences of two cell wall proteins of Saccharomyces cerevisiae, encoded by the genes YRM305C and YGR279C. However, the antigenic peptides were those showing the least similarity with the corresponding regions of the above proteins. In particular, the lymphoproliferation-inducing sequence of the T1 peptide scored only 20% identity with the homologous regions of S. cerevisiae proteins. Besides disclosing the amino acid sequence of MP65, this study provides an initial characterization of some of its antigenic determinants, as well as of synthetic peptides of potential use to detect specific immune responses against MP65, a major target of anticandidal cell-mediated immunity in humans.     

Rapid screening for early detection of mother-to-child transmission of human immunodeficiency virus type 1.

The testing of dried blood spots (DBSs) for the presence of human immunodeficiency type 1 (HIV-1) proviral DNA by PCR was first described in 1991. The technology has proven to be particularly valuable for resolving the infection status in HIV-1-indeterminate infants born to HIV-1-seropositive mothers. To broaden the applicability of DBS PCR, we adapted it to a standardized, commercially available microwell plate amplification and detection kit, Amplicor HIV-1, produced by Roche Diagnostic Systems. The microwell assay is rapid and easy to perform and uses equipment that is readily available in routine diagnostic laboratories. The high level of performance of the assay was demonstrated in 1,168 duplicate tests performed on 584 DBSs from 178 uninfected and 100 HIV-1-infected individuals, including 56 children with perinatally acquired HIV-1. Of 12 infants who were followed prospectively from birth, 3 (25%) were infected in utero (PCR positive at birth) and 9 (75%) were infected intrapartum (PCR negative, culture negative at birth). Overall, HIV-1 DNA was identified in 3 of 11 (27.3%) DBSs collected from infected infants during the first 4 days of life, 8 of 9 (88.9%) DBSs collected between 10 and 15 days postpartum, and 166 of 167 (99.4%) DBSs collected after 15 days of age. All 320 DBSs from uninfected children were PCR DNA negative. These findings indicate that use of the Roche microwell DBS PCR assay provides a powerful new approach for large-scale perinatal screening programs and population-based studies of vertical transmission.     

A new approach to the design of internal fixation plates

Mathematical analysis by finite element modeling methods was used in conjunction with laboratory bench experiments in selecting appropriate stiffness parameters for internal fixation plate designs. Bench experiments performed included tests of an idealized plated magnesium tube and of a plated canine femur. Finite element modeling of the plate-tube and plate-bone structures was also performed, and the computed results were compared with those obtained experimentally. Three types of internal fixation plates--one "rigid" control plate and two less rigid experimental plates--were examined using both the finite element and experimental models. These plates were further examined by finite element modeling of a plated human femur during single stance loading. In all cases, the "rigid" control plate was found to shield the underlying bone from stress, while both experimental plates were found to have significantly less bone stress shielding. Our findings suggest that an improved plate design should have a low axial stiffness but moderate bending and torsional stiffnesses to facilitate fracture healing and bone remodeling without causing osteopenia.     

Scrotal carcinoid. Presenting manifestation of multiple lesions in the small intestine

A middle-aged man presented with a paratesticular carcinoid tumor, his first manifestation of multiple carcinoid tumors of the small bowel. Of the nine carcinoids reported in the English-language literature as metastatic to the scrotum, five simulated a primary lesion. Although the bulk of scrotal carcinoids arise in the testis, the differential diagnosis always should include metastasis from an extrascrotal source.     

Recognition of extracellular matrix proteins by Paracoccidioides brasiliensis yeast cells.

The adhesion of microorganism to host cells or extracellular matrix (ECM) proteins is the first step in the establishment of an infectious process. Interaction between Paracoccidioides brasiliensis yeast cells and ECM proteins has been previously noted. In vivo, in the chronic phase of experimental paracoccidioidomycosis (PCM), laminin and fibronectin have been detected on the surface of yeast cells located inside granulomatous lesions. The aim of the present study was to examine the ability of P. brasiliensis yeast cells to interact with extracellular matrix proteins (laminin, fibrinogen and fibronectin) and to establish which molecules were involved in this interaction. Immunofluorescence microscopy and flow cytometry demonstrated that all three ECM proteins tested were able to bind to the surface of P. brasiliensis yeast cells. Treatment with trypsin, chymotrypsin, chitinase, proteinase K or different sugars resulted in no change in laminin binding. In addition, ligand affinity assays were performed using different yeast extracts (total homogenates, beta-mercaptoethanol, SDS extracts). These assays demonstrated the presence of 19 and 32-kDa proteins in the cell wall with the ability to bind to laminin, fibrinogen and fibronectin. This interaction could be important in mediating attachment of the fungus to host tissues and may consequently play a role in the pathogenesis of PCM.     

Dynamin 2 regulates T cell activation by controlling actin polymerization at the immunological synapse

Actin reorganization at the immunological synapse is required for the amplification and generation of a functional immune response. Using small interfering RNA, we show here that dynamin 2 (Dyn2), a large GTPase involved in receptor-mediated internalization, did not alter antibody-mediated T cell receptor internalization but considerably affected T cell receptor-stimulated T cell activation by regulating multiple biochemical signaling pathways and the accumulation of F-actin at the immunological synapse. Moreover, Dyn2 interacted directly with the Rho family guanine nucleotide exchange factor Vav1, and this interaction was required for T cell activation. These data identify a functionally important interaction between Dyn2 and Vav1 that regulates actin reorganization and multiple signaling pathways in T lymphocytes.     

Tertiary telemedicine support during global military humanitarian missions.

OBJECTIVE: The U.S. military medical units face the challenge of delivering the latest medical knowledge and advances to remote areas of the world. This study details the tertiary care telemedicine support by Walter Reed Army Medical Center (WRAMC) for these humanitarian missions in several locations worldwide. METHODS: This study encompasses data from all clinical consults received at WRAMC, from the earliest deployment of a satellite-based system in February 1993 through February 1996. The analysis of the consultations include type of consult, medical specialty consulted, response time, and technology used. RESULTS: Two hundred forty consults were received from 12 remote telemedicine sites supporting military medical missions. The consults used a combination of "store-and-forward" technologies and interactive video conferencing systems. Establishment of a telemedicine service at WRAMC, including medical and technical support personnel, facilitated the delivery of telemedicine consultations. Several concurrent missions were supported. The distribution of consults included medicine (40%), surgery (36%), radiology (21%), and dentistry (3%). The most frequently consulted medical subspecialty was dermatology (29%) followed by orthopedic surgery (16%). Most consults were routine (88%). Ninety-four percent of the consults were completed within the predefined telemedicine response criteria (24 hours for routine consults and 3 hours for emergencies). CONCLUSION: The study illustrates the importance of a responsive telemedicine service at a tertiary facility supporting simultaneous medical missions. Access to the complete spectrum of medical and surgical specialties was essential to enhancing the delivery of medical care. The critical analysis and evaluation of this experience will assist in designing future prospective studies for evaluation of telemedicine.     

SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2

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