Methicillin-resistant and methicillin-susceptible Staphylococcus aureus in dairy sheep and in-contact humans: An intra-farm study.

Staphylococcus aureus is involved in a wide variety of diseases in humans and animals, and it is considered one of the most significant etiological agents of intramammary infection in dairy ruminants, causing both clinical and subclinical infections. In this study, the intra-farm prevalence and circulation of methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) were investigated on an Italian dairy sheep farm previously identified as MRSA-positive by testing bulk tank milk (first isolation in 2012). Human samples (nasal swabs, hand skin samples, and oropharyngeal swabs) from 3 persons working in close contact with the animals were also collected, and the genetic characteristics and relatedness of the MRSA isolates from human and animal sources within the farm were investigated. After 2yr from the first isolation, we confirmed the presence of the same multidrug-resistant strain of MRSA sequence type (ST)1, clonal complex (CC)1, spa type t127, staphylococcal cassette chromosome mec (SCCmec) type IVa, showing identical pulsed field gel electrophoresis (PFGE) and resistance profiles at the farm level in bulk tank milk. Methicillin-resistant S. aureus isolates were detected in 2 out of 556 (0.34%) individual milk samples, whereas MSSA isolates were detected in 10 samples (1.8%). The MRSA were further isolated from udder skin samples from the 2 animals that were MRSA-positive in milk and in 2 of the 3 examined farm personnel. All MRSA isolates from both ovine and human samples belonged to ST(CC)1, spa type t127, SCCmec type IVa, with some isolates from animals harboring genes considered markers of human adaptation. In contrast, all MSSA isolates belonged to ruminant-associated CC130, ST700, spa type t528. Analysis by PFGE performed on selected MRSA isolates of human and animal origin identified 2 closely related (96.3% similarity) pulsotypes, displaying only minimal differences in gene profiles (e.g., presence of the immune evasion cluster genes). Although we observed low MRSA intra-farm prevalence, our findings highlight the importance of considering the possible zoonotic potential of CC1 livestock-associated MRSA, in view of the ability to persist over years at the farm level. Biosecurity measures and good hygiene practices could be useful to prevent MRSA spread at the farm level and to minimize exposure in the community and in categories related to farm animal industry (e.g., veterinarians, farmers, and farm workers).

Case report: Chalazion and its features visualized by ultrahigh resolution optical coherence tomography.

PURPOSE: The purpose of this case report is to highlight the clinical characteristics of a recurrent chalazion through the use of digital photography and ultra-high resolution optical coherence tomography (UHROCT). CASE REPORT: A single case is presented, along with digital biomicroscopic photographs and UHROCT images. DISCUSSION: A review of the literature describing the histopathological and associations of chalazia and other disorders, suggest it may be possible to differentiate different eyelid conditions based on their clinical manifestations and appearance on UHROCT tomograms. Based on the images presented here, it appears that this case is typical of a post-menopausal incidence of chalazion and risk for acne rosacea.

Contact lens induced papillary conjunctivitis with silicone hydrogel lenses.

PURPOSE: To describe the refitting of a soft lens wearer into a silicone hydrogel lens due to neovascularization. This change, in turn, caused contact lens induced papillary conjunctivitis (CLPC) and a further refitting was necessary. METHODS: The patient was refit into a high Dk surface treated silicone hydrogel with a high modulus value. A second refitting was undertaken into a lower Dk silicone hydrogel contact lens with a lower modulus value which had no surface treatment but incorporated an internal wetting agent. RESULTS: A high Dk/t lens was used to resolve existing neovascularization and chronic hyperaemia. Subsequently, CLPC response occurred, possibly due to a combination of factors, resulting in irritation of the palpebral conjunctiva. This resulted in temporary lens discontinuation. A second silicone hydrogel lens was fit, along with the use of a non-preserved care system, which led to improvement and eventual resolution of the condition. CONCLUSION: High Dk silicone hydrogel lenses have shown excellent efficacy in resolving hypoxic complications such as neovascularization and hyperaemia. However, attention needs to be paid to their potential effect on the upper tarsal plate. More than one silicone hydrogel lens may be needed to help resolve these issues.

Prevalence and concentration of Verotoxigenic Escherichia coli O157:H7 in adult sheep at slaughter from Italy.

A 1-year study on the animal-level prevalence and concentration of Escherichia coli O157 in adult sheep at slaughter was performed, to collect qualitative and quantitative information on the diffusion of the pathogen in adult sheep from Italy. A total 533 samples were collected, with a similar distribution in the four seasons. For prevalence estimates, a simple random sampling technique was used. An immuno-magnetic separation technique was used for sample screening, with enumeration of the pathogen in positive samples, along with molecular and serological identification of isolates. An overall prevalence of 7.1% (38/ 533, 95% CI 4.9-9.3%) was observed for fully virulent E. coli O157. A wide interval of VTEC O157 per gram was observed (< 100 to 6 x 10(5) CFU g(-1)), with 28.9% (11/38) of positive samples > or = 1 x 10(3) CFU g(-1), set as the threshold for those animals defined 'active shedders' for the purpose of the study. Eight per cent (3/38) of animals shed > 1 x 10(4) g(-1) VTEC O157, which represents > 96% of the total VTEC O157 bacteria cultured from all animals tested. The prevalence estimate of active shedders was therefore 2.1% (95% CI 0.9-3.3%). Most (34/38, 89.5%) of the positive animals were found in summer (July-September). Prevalence and concentrations of virulent VTEC O157 obtained in this study contribute to the demonstration that adult sheep represent a relevant source of environmental contamination from virulent VTEC O157, as well as a source of VTEC O157 contamination for food of ovine origin (meat and dairy products), especially during warm months.

Asymptomatic corneal staining associated with the use of balafilcon silicone-hydrogel contact lenses disinfected with a polyaminopropyl biguanide-preserved care regimen.

PURPOSE: To compare subjective symptoms and signs in a group of individuals who wear silicone-hydrogel lenses on a daily wear basis while they sequentially used two differing care regimens. METHODS: Fifty adapted soft-lens wearers were fitted with a silicone-hydrogel lens material (PureVision, Bausch & Lomb). The lenses were worn on a daily wear basis for two consecutive 1-month periods, during which the subjects used either a Polyquad (polyquaternium-1) -based system or a polyaminopropyl biguanide (PHMB) -based system, using a double-masked, randomized, crossover experimental design. RESULTS: Significant levels of relatively asymptomatic corneal staining were observed when subjects used the PHMB-based system, with 37% of subjects demonstrating a level of staining consistent with a classical solution-based toxicity reaction. Only 2% of the subjects exhibited such staining when using the Polyquad-based system. These results were significantly different (p < 0.001). Significant symptoms were not correlated with the degree of staining, with no differences in lens comfort or overall preference being reported between the regimens (p = NS). The only statistically significant difference in symptoms related to minor differences in stinging after lens insertion being reported, with the Polyquad-based system demonstrating less stinging (p < 0.008). CONCLUSIONS: Practitioners who fit silicone-hydrogel contact lenses on a daily wear basis should be wary of the potential for certain PHMB-containing multipurpose care systems to invoke corneal staining. Switching to non-PHMB based regimens will eliminate this complication in most instances.

Cutaneous lymphomatoid granulomatosis: correlation of clinical and biologic features.

Lymphomatoid granulomatosis (LYG) is a rare angiocentric and angiodestructive Epstein-Barr virus-associated B-cell lymphoproliferative disorder (EBV-BLPD), varying widely from an indolent process to an aggressive large cell lymphoma. The skin is the extrapulmonary organ most commonly involved in LYG. We studied 32 skin lesions from 20 patients with known pulmonary LYG, using immunohistochemistry, in situ hybridization for EBV, and polymerase chain reaction for the presence of antigen receptor gene rearrangements (IgH and TCR) to better define both the clinicopathologic spectrum and pathogenesis of the cutaneous lesions. We describe two distinct patterns of cutaneous involvement. Multiple erythematous dermal papules and/or subcutaneous nodules, with or without ulceration, were present in 17 patients (85%). These lesions demonstrate a marked angiocentric lymphohistiocytic infiltrate, composed predominantly of CD4-positive T-cells, with a high propensity for involving the subcutaneous tissues, and exhibiting angiodestruction, necrosis, and cytologic atypia. EBV-positive B-cells were detected in the nodules from five patients; clonal immunoglobulin heavy chain gene (IgH) rearrangements were detected by polymerase chain reaction in two patients. Multiple indurated, erythematous to white plaques were present in three patients (15%). The plaque lesions were negative for EBV and clonal IgH gene rearrangements in all cases studied. The clinical course of overall disease was variable, ranging from spontaneous regression without treatment (1 of 13; 7%), resolution with chemo/immunomodulatory therapy (8 of 13; 62%), and progression (4 of 13; 31%). The clinical and histopathologic features of cutaneous LYG are extremely diverse. However, the majority (85%) of the cutaneous lesions mirrors to some extent LYG in the lung, although EBV+ cells are less frequently identified. This subset of cases shows the histopathologic triad of angiodestruction with associated necrosis, panniculitis, and in some cases atypical lymphoid cells. The commonality of the histologic features in this group suggests a common pathophysiologic basis, possibly mediated by cytokines and chemokines induced by EBV. A small percentage of the lesions (15%) presented as indurated and atrophic plaques, and EBV was not identified in the small number of cases studied. The relationship of the plaque-like lesions to LYG remains uncertain. Whereas some cases of LYG regress spontaneously, most require therapy.

Regression and clonally distinct recurrence of human immunodeficiency virus related Burkitt-like lymphoma during antiretroviral therapy.

An increased incidence of intermediate to high-grade Non Hodgkin's Lymphoma is found in individuals with AIDS. Although immune function in AIDS patients can be improved through the use of antiretroviral therapy, the contribution of these drugs to lymphoma regression is not known. Here we describe the complete regression and subsequent recurrence of high grade, Burkitt-like lymphoma during antiretroviral therapy in a patient with AIDS. Antiretroviral therapy resulted in diminished viral load and modest improvement in CD4+ T cell counts. Lymphoma regressed initially, but relapsed 3 months later. Tissue taken from the initial and recurrent tumor demonstrated different clonal rearrangements. The recurrent lymphoma did not respond to continued antiretroviral therapy. In Conclusion, antiretroviral therapy may contribute to lymphoma regression in AIDS lymphoma. Clinically recurrent disease may be clonally distinct.

Chemokine gene expression and clonal analysis of B cells in tissues involved by lymphoid interstitial pneumonitis from HIV-infected pediatric patients.

Lymphoid interstitial pneumonitis (LIP), a frequent pulmonary complication in HIV-infected pediatric patients, is characterized histologically by marked infiltration of lymphoid cells. We sought to evaluate the nature and pathogenesis of the lymphoid infiltrates and to examine the relationship of LIP to pulmonary MALT lymphoma that has been described in pediatric HIV positive patients. To examine the potential contribution of chemokines and cytokines to the inflammatory cell recruitment in tissues involved by lymphoid interstitial pneumonitis from HIV-infected pediatric patients, RNA was extracted from paraffin-embedded tissues from five lung biopsies in four pediatric HIV-positive patients and from five control, normal lung biopsies in five HIV-negative patients and was analyzed by semiquantitative RT-PCR for the expression of cytokines (TNF-alpha, GM-CSF, IFN-gamma, IL-4, IL-6, IL-10, and IL-18) and chemokines (IP-10, Mig, regulated upon activation, normal T expressed and secreted [RANTES], and MIP1-alpha and beta) after normalization for G3PDH. Expression of IL-18 was increased, as well as expression of IFN-gamma-inducible chemokines IP-10 and Mig in LIP tissues compared with controls. RANTES and MIP1-alpha and -beta were also increased in pediatric LIP lesions compared with controls. In contrast, expression of TNF-alpha, GM-CSF, IL-10, and IL-6 was variable in LIP tissues and controls. In addition, clonality of the B-cell population was evaluated by VDJ-PCR. A polyclonal B-cell population was shown in all five biopsies from five patients with LIP; and in one patient with concurrent LIP and MALT lymphoma, a band of increased intensity was observed in the LIP biopsy that was identical in size to the monoclonal band in the concurrent MALT lymphoma biopsy. These results provide evidence of high-level expression of certain chemokines in lymphoid interstitial pneumonitis tissues and suggest that chemokines and cytokines may play an important role in the recruitment of inflammatory cell infiltrates into these tissues. In addition, LIP may represent an early stage of MALT lymphoma or an immunologic response to a chronic antigenic stimulus that may provide a milieu or microenvironment for the evolution of a monoclonal B-cell population.

Peripheral T-cell lymphoma with aberrant expression of CD79a and CD20: a diagnostic pitfall.

Immunohistochemical studies are increasingly used for the routine diagnosis of lymphomas as it is widely accepted that lymphomas of different cell lineages vary in their prognosis and response to therapy. A case of peripheral T-cell lymphoma with aberrant expression of B-cell-associated antigens L-26 (CD20) and mb-1 (CD 79a) is described. The disease pursued an aggressive clinical course, and the patient died of disease 6 weeks after presentation. Immunohistochemical studies demonstrated expression of both T- and B-cell-associated antigens, including CD3, CD8, CD43, TIA-1, CD20, and CD79a. Other markers expressed by the tumor cells included CD56 and S-100. Of interest, betaF-1 staining for the beta chain of T-cell receptor (TCR) complex was positive in the small admixed T lymphocytes but was negative in the tumor cells, raising the possibility of a gamma/delta T-cell lymphoma. Molecular studies by polymerase chain reaction (PCR) demonstrated clonal TCR-gamma chain gene rearrangement without evidence for a clonal rearrangement of the immunoglobulin heavy chain gene. PCR for HHV-8 related sequences was negative. Mb-1 is an IgM-associated protein that was thought to be restricted to normal and neoplastic B cells. Although its coexpression has been reported in up to 10% cases of precursor T-cell lymphoblastic lymphoma, the coexpression of both CD20 and CD79a has not been described in mature T-cell malignancies. Biphenotypic lymphomas associated with HHV-8 have been reported in immunodeficiency, but no evidence of immune deficiency was identified, and studies for EBV and HHV-8 were negative. This case illustrates that no marker has absolute lineage specificity and that immunophenotypic studies should always be performed with panels of monoclonal antibodies. Moreover, cases with ambiguous phenotypes may require genotypic studies for precise lineage assignment.

Large granular lymphocyte leukaemia is characterized by a clonal T-cell receptor rearrangement in both memory and effector CD8(+) lymphocyte populations.

Large granular lymphocyte (LGL) leukaemia is a disease with increased numbers of circulating granular lymphocytes and an increased percentage of clonally rearranged CD8(+)CD57(+) cells. To determine whether LGL cells are also found in other lymphocyte subsets, CD8(+) cells from 10 LGL patients were sorted into CD57(+) and CD57(-) fractions and analysed for clonality using a T-cell receptor gamma (TCR gamma) polymerase chain reaction (PCR). In nine patients, a clonal TCR rearrangement was identified in the CD8(+)CD57(+) cells, and in one patient, the TCR rearrangement was oligoclonal in the CD8(+)CD57(+) fraction. In eight out of nine of the clonally rearranged patients, the same band was also present in the CD8(+)CD57(-) fraction. To define the relationship between CD57(-) and CD57(+) LGL populations, CD8(+)CD57(-) and CD8(+)CD57(+) cells were sorted from five patients and cultured in the presence of anti-CD3 plus CD28 antibodies. The CD57(+) cells died of apoptosis before d 7, while the CD57(-) cells proliferated and differentiated into CD57(+) cells. Clonal analysis identified the same band in both cultured subpopulations and in the uncultured CD8(+) cells. Immunophenotypical analysis showed that CD8(+)CD57(-) cells expressed memory cell markers, while the CD8(+)CD57(+) cells exhibited effector characteristics. These results suggest that LGL disease originates in a CD57(-) memory T-cell compartment that continually generates CD57(+) (effector cell) progeny.

Coincident myelodysplastic syndrome and T-cell large granular lymphocytic disease: clinical and pathophysiological features.

Myelodysplastic syndrome (MDS) and T-cell large granular lymphocytic disease (T-LGL) are bone marrow failure disorders. Successful use of immunosuppressive agents to treat cytopenia in MDS and LGL suggests a common pathophysiology for the two conditions. Of 100 patients with initial diagnoses of either MDS or T-LGL referred to the National Institutes of Health for immunosuppressive treatment of cytopenia, nine had characteristics of both T-LGL and MDS (T-LGL/MDS). Fifteen patients with T-LGL received cyclosporin (CSA) (10 responses). Eight out of nine patients with T-LGL/MDS received CSA (two responses) and one patient received ATG (one response). Of 76 patients with MDS, eight received CSA (one response) and 68 received ATG (21 responses). The response to immunosuppression was significantly lower in patients with T-LGL/MDS and MDS than in patients with T-LGL disease alone (28% vs. 66%, P = 0.01). The proportion of T-helper cells and T-suppressor cells with an activated phenotype (HLA-DR(+)) was increased in patients with T-LGL, T-LGL/MDS and MDS, but the increase in activated T-suppressor cells in patients with T-LGL/MDS was not statistically significant. Autoreactive T cells may suppress haematopoiesis and contribute to the cytopenia in T-LGL and some patients with MDS, leading to T-LGL/MDS. The lower response rate of MDS or T-LGL/MDS to immunosuppression, compared with T-LGL alone, may reflect the older age and intrinsic stem cell abnormalities in MDS and T-LGL/MDS patients.

Alcohol, smoking, coffee and risk of non-fatal acute myocardial infarction in Italy.

OBJECTIVES: To assess the relation between cigarette smoking, alcohol, coffee, decaffeinated coffee and tea consumption, and the risk of non-fatal acute myocardial infarction (AMI). DESIGN AND SETTING: Hospital-based case-control study conducted in 1995-1999 in Milan, Italy. PATIENTS: 507 cases with a first episode of non-fatal AMI, and 478 controls admitted to hospital for acute diseases. METHODS: Information was collected by interviewer-administered questionnaires. Odds ratios (OR) and 95% confidence intervals (CI) were calculated by multiple logistic regression. RESULTS: Compared to alcohol non-drinkers the OR was 0.6 (95% CI: 0.4-0.9) in drinkers, and 0.5 in drinkers of > 3 drinks/day. The OR for > 1 drink/day of wine was 0.5, and those for beer, amari, grappa and spirits ranged between 0.4 and 0.6. Compared to never smokers, the OR was 2.2 (95% CI: 1.5-3.1) among current smokers, and 4.6 among current smokers of > or = 25 cigarettes/day. The risk was similar to that of never smokers > or = 5 years after cessation (OR: 1.1 after 5-9 years, 0.7 after > or = 10 years). The OR was 2.3 for low tar cigarettes and 2.0 for high tar ones. The OR for coffee intake (expresso and mocha) was around unity up to 3 cups/ day, but rose to 1.9 (95% CI: 1.1-3.3) for > or = 6 cups/ day. Moderate decaffeinated coffee and tea intake was not associated with AMI risk. Compared to non-smokers drinking < or = 3 cups of coffee/day, the OR was 1.6 among non-smokers drinking > 3 cups of coffee/ day and 3.3 (95% CI: 2.1-5.0) among current smokers drinking < or = 3 cups of coffee/day. Compared to alcohol drinkers with a coffee intake of < or = 3 cups/ day, alcohol non-drinkers with higher coffee intake had an OR of 2.2, and compared to non-smokers alcohol drinkers, the OR was 3.3 in current smokers alcohol non-drinkers. CONCLUSIONS: In this Italian population alcohol intake was inversely associated to AMI risk, while smoking and heavy (but not moderate) coffee drinking increased the risk.

Molecular confirmation of Ewing sarcoma.

OBJECTIVE: To analyze retrospectively results of reverse transcription polymerase chain reaction (RT-PCR) testing and demographic information in patients with known or suspected Ewing sarcoma/primitive neuroectodermal tumor family of tumors referred to the National Cancer Institute and to describe factors influencing the determination of molecular marker status. PATIENTS AND METHODS: Tumor samples from 76 patients from February 1997 to December 1999 were analyzed. In all cases, the diagnosis of this family of tumors was confirmed by histopathologic review. RESULTS: In 58 patients, the presence of a translocation associated with this family of tumors was confirmed using RT-PCR. Specifically, there were 45 Ewing sarcoma (EWS)-FLI type 1 translocations, four EWS-FLI type 2 translocations, five EWS-ERG translocations, and four less common EWS-FLI variants. Of patients with a confirmed translocation, four were confirmed only after nested RT-PCR techniques were used. In five patients who initially underwent needle biopsy, the diagnosis was confirmed only after open biopsy or repeat needle biopsy was undertaken. Samples from 18 patients were translocation-negative. Of these, seven samples were deemed inadequate for RT-PCR testing as a result of inappropriate tissue handling or the presence of necrotic material. Five patients were found to have a different diagnosis after complete histopathologic and molecular characterization. Six samples remained, in which adequate tissue was obtained with no evidence of a characteristic translocation. CONCLUSIONS: In apparently translocation-negative samples, close attention should be given to the possibility of an alternative diagnosis, the potential need for nested RT-PCR, and the possibility of an inadequate sample. Strong consideration should be given to the use of open biopsy as opposed to needle biopsy to avoid the need for repeat biopsies and the potential for inaccurate assessment of molecular marker status.

gamma delta T-cell lymphoma of the skin: a clinical, microscopic, and molecular study.

BACKGROUND: Only a few cases of primary gamma delta cutaneous T-cell lymphoma (CTCL) have been reported. We encountered 3 cases of this rare condition. OBJECTIVES: To characterize gamma delta CTCL by clinical, microscopic, and molecular methods and to investigate the role of Epstein-Barr virus (EBV) infection in its pathogenesis. DESIGN: Patients were evaluated by clinical examination, and biopsy specimens of lesional skin were examined by light microscopy and immunohistochemistry. Polymerase chain reaction amplification for T-cell receptor gamma gene rearrangements and in situ hybridization for EBV were performed on 3 biopsy specimens. SETTING: National Institutes of Health, a tertiary referral center. PATIENTS: Individuals with a clinical and histologic diagnosis of primary gamma delta CTCL. OUTCOME MEASURES: Clinical, light microscopic, and immunohistochemical features, and the presence of T-cell rearrangement and EBV RNA in biopsy specimens. RESULTS: Patients exhibited multiple plaques, tumors, and/or subcutaneous nodules primarily distributed over the extremities. Individuals exhibited an aggressive clinical course with resistance to multiagent chemotherapy and radiation. Microscopic examination revealed epidermotropism in 2 cases, a dermal infiltrate in all 3 cases, and subcutaneous involvement in 1 case. Immunohistochemical studies showed the presence of CD3(+)TCR delta(+) in 3 patients, CD8(+)in 1, and CD4(+), CD20(+), CD56(+), and beta F1(+) in none. All 3 cases exhibited an activated cytotoxic T-cell phenotype positive for T-cell intracellular antigen 1, perforin, and granzyme B. A clonal T-cell receptor gamma chain gene rearrangement was detected in all 3 cases by polymerase chain reaction. In situ hybridization was negative for EBV sequences in all 3 cases. CONCLUSION: gamma delta Cutaneous T-cell lymphomas are EBV-negative lymphomas that express a mature cytotoxic phenotype and have an aggressive clinical behavior. Arch Dermatol. 2000;136:1024-1032

Fulminant EBV(+) T-cell lymphoproliferative disorder following acute/chronic EBV infection: a distinct clinicopathologic syndrome.

This study describes the clinicopathologic features of 5 patients who developed a fulminant Epstein-Barr virus (EBV)-positive clonal T-cell lymphoproliferative disorder (LPD) after acute EBV infection. One additional patient developed a similar disorder in the setting of long-standing chronic active EBV infection. Detailed immunophenotyping, in situ hybridization for EBV early RNA-1 (EBER1) and polymerase chain reaction (PCR) analyses for immunoglobulin (Ig) heavy chain and T-cell receptor (TCR)-gamma gene rearrangements were performed on paraffin-embedded tissue from all patients. In addition, EBV strain typing and detection of the characteristic 30-bp deletion of the latent membrane protein-1 (LMP-1) gene were performed by PCR. Controls included 8 cases of uncomplicated infectious mononucleosis (IM). Patients included 4 males and 2 females with a median age of 18 years (2-37 years). Three patients were Mexican, 2 were white, and 1 was of Asian descent. All presented with fever, hepatosplenomegaly, and pancytopenia; 5 were previously healthy, but had a clinical history of a recent viral-like upper respiratory illness (1 week to 2 months), and 1 patient had documented chronic active EBV infection for 7 years. Serologic data for EBV were incomplete but titers were either negative or only modestly elevated in 3 cases. In 1 case serology was consistent with severe chronic active EBV infection. In the remaining 2 cases serologic studies were not performed. All patients died within 7 days to 8 months of presentation with T-cell LPD. On histologic examination, the liver and spleen showed prominent sinusoidal and portal lymphoid infiltrates of CD3(+), beta F1(+), EBER1(+) T cells lacking significant cytologic atypia. Two cases were CD4(+), 2 cases were CD8(+), and 2 cases had admixed CD4(+) and CD8(+) cells without clear subset predominance. All were TIA-1(+), CD56(-). Only rare B cells were noted. Marked erythrophagocytosis was present. Molecular analysis revealed identical T-cell clones in 2 or more sites (liver, spleen, lymph node) in 5 cases. All patients carried type A EBV; 4 cases had wild-type EBV-LMP, and 2 showed the 30-bp deletion. This fulminant T-cell LPD after acute/chronic EBV infection is characterized by hepatosplenomegaly, often without significant lymphadenopathy, fever, liver failure, pancytopenia, and erythrophagocytosis indicative of a hemophagocytic syndrome. EBV serology may be misleading, with lack of elevated titers. The presence of an EBER1(+) T-cell infiltrate with scant B cells should alert one to this diagnosis. Although cytologic atypia is minimal, studies for T-cell clonality confirm the diagnosis. (Blood. 2000;96:443-451)

Association of EBV strains, defined by multiple loci analyses, in non-Hodgkin lymphomas and reactive tissues from HIV positive and HIV negative patients.

Epstein-Barr virus (EBV) associated with lymphoid neoplasms demonstrates preferential association with certain viral strains. Previous subtyping studies have however been confined to analysis of sequence variability within a single locus in EBV. Variations have now been reported for several latently expressed EBV genes, including, EBNAs-1, 2 and LMP-1. Variant EBNA-1 strains have been identified in Burkitt's lymphomas and clustering of subtypes for LMP and EBNA-2 have been associated with either malignancy and/or clinical disease. To investigate the linkage between the variability in these three loci in EBV associated with lymphoid malignancies, we subclassified EBV-associated lymphoproliferations (9 reactive and 24 malignant) from HIV-negative and HIV-positive patients by analysis of the EBNA-1, LMP1, and EBNA-2 genes. Our results demonstrate that (1) EBV identical to the prototype B95.8 strain (Type 1 EBNA-2, wild type EBNA-1 and LMP-1) is very rarely associated with tumors. (2) The EBNA-1 variant V-leucine, restricted to malignant lymphomas in immunocompetent patients, was readily identified in non-malignant lesions in HIV infected patients. (3) Variations of EBNA-1 occur independent of variations at other loci.

Development of lymphoproliferative disorder of granular lymphocytes in association with hairy cell leukemia.

Lymphoproliferative disorder of granular lymphocytes (LDGL) is a low grade T-cell disease characterized by clonal expansion of large granular lymphocytes of either T cell or natural killer (NK) cell lineage that express the cytotoxic T-cell/NK cell antigens CD16, CD56 and/or CD57. LDGL has been described in association with other malignancies, leading to theories of a common abnormal stem cell as well as development of the LDGL as an immune response to a primary tumor. We have studied 32 patients with hairy cell leukemia (HCL). In 15 patients (47%) we detected an increase in cells expressing cytotoxic T-cell/NK cell antigens. In 10(31%) patients these cells were of T cell lineage, while 5 patients (16%) had increased NK-cells. T cell clonality was detected by PCR in all cases with increased cytotoxic T-cells in which adequate DNA was obtained from peripheral blood. Since in 2 patients the LDGL was not present at diagnosis but developed during follow up, our data suggests that clonal LDGL may develop in response to the HCL. The significance of LDGL in the setting of HCL and flow cytometric evaluation of HCL versus LDGL will be discussed.

Modulation of clinical drug resistance in a B cell lymphoma patient by the protein kinase inhibitor 7-hydroxystaurosporine: presentation of a novel therapeutic paradigm.

Emerging evidence suggests that apoptosis is an important mechanism of tumor cell death from antineoplastic therapy. 7-hydroxystaurosporine (UCN-01) is a novel protein kinase inhibitor that increases chemotherapy-induced apoptosis in vitro and is in early phases of clinical development. In this report, we present a 68-year-old patient with chemotherapy-resistant lymphoma treated with UCN-01 and chemotherapy. He had a stage IV plasmacytoid lymphoma that failed to enter remission with high-dose EPOCH II (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin) chemotherapy. Due to disease progression and transformation to large cell lymphoma in the liver and bone marrow, he received UCN-01. Four weeks later, he received "standard-dose" EPOCH because of progression, developed severe neutropenia for 9 days, and expired from Candida sepsis on day 23. At autopsy, there was no histological evidence of residual lymphoma, although PCR for immunoglobulin gene rearrangement analysis revealed a faint clonal band in two of six nodes but none in the liver. Significantly, no B cells were detected by immunohistochemistry in lymph nodes, and a polyclonal ladder pattern associated with the presence of normal B cells was not seen in the immunoglobulin gene rearrangement PCR assay. Profound peripheral lymphopenia (50 cells/microliter) was also observed. Pharmacokinetics showed UCN-01 salivary concentrations, a surrogate for free drug concentrations, to be within an effective range in vitro (45 nmol/L) as a modulator of DNA-damaging agent cytotoxicity. In vitro, UCN-01 is synergistic with multiple cytotoxic agents and increases fludarabine-induced apoptosis in a human breast cell line. These results suggest that UCN-01 sensitized the lymphoma to the cytotoxic effects of EPOCH, possibly by modulating the "threshold" for apoptosis, and may illustrate a new paradigm for reversal of drug resistance.

Epstein-Barr virus-positive primary gastrointestinal Hodgkin's disease: association with inflammatory bowel disease and immunosuppression.

Inflammatory bowel disease (IBD) is associated with an increased risk of lymphoma, which is usually extraintestinal but sometimes may involve the diseased bowel itself. Most lymphomas described in this setting are of non-Hodgkin's type, but rare cases of Hodgkin's disease (HD) have been reported. We describe the clinicopathologic and molecular features of four patients with primary gastrointestinal HD. Three patients had preexistent Crohn's disease (CD), for which two of them had received immunosuppressive therapy. The fourth patient had a longstanding history of diverticulitis and myasthenia gravis and was receiving immunosuppressive therapy for the latter. Multifocal involvement of the bowel by HD was noted in all four cases. Disease was staged as IVA in one patient, IIIB in one patient, and IE in one patient, and the fourth patient died in the postoperative period before further workup. Two patients received chemotherapy, one of whom was dead at 9 months, whereas the other has no evidence of disease at 25 months' follow-up. The patient with IE disease did not receive any therapy because only a few microscopic foci of disease were present and is also without any evidence of disease at 17 months. The Reed-Sternberg (RS) cells in all four cases expressed CD30, CD15, EBER-1, and LMP-1; two of four were focally CD20-positive. VJ-polymerase chain reaction for immunoglobulin heavy chain (IgH) rearrangement showed a polyclonal pattern in all four cases. In two cases, laser capture microdissection was used to isolate individual RS and Hodgkin's cells, which contained rearranged immunoglobulin genes, confirming a B-cell genotype. Whereas one case showed a dominant clonal band present in all isolates, cells from the patient with stage IE disease clearly showed a polyclonal population of RS cells. Our findings indicate that HD arising in the setting of IBD or chronic inflammation is the result of an Epstein-Barr virus-driven lymphoproliferation, analogous to that found in other immunodeficient states. Disordered immunoregulation inherent to CD and immunosuppressive therapy for the latter may contribute to its development. The finding of polyclonal RS cells in a patient with early stage disease and apparent cure by surgical resection versus monoclonal RS cells in the patient with disseminated disease suggests that HD in the setting of immunodeficiency also may show molecular progression, in a manner similar to that occurring in conventional B-cell lymphoproliferative disorders arising in the same setting.

Microcystic adnexal carcinoma associated with primary immunodeficiency, recurrent diffuse herpes simplex virus infection, and cutaneous T-cell lymphoma.

Cutaneous microcystic adnexal carcinoma (MAC) is a rare and poorly understood tumor that predominantly occurs in the head and neck. MAC usually affects people in their fourth and fifth decades. Some patients have had a history of radiation. We present a case of MAC occurring in the left antecubital fossa of an 18-year-old white woman with an unusual immunodeficiency syndrome. The patient also developed a squamous cell carcinoma, a cutaneous T-cell malignancy, and a perigastric leiomyoma. A congenital infection of herpes simplex virus (HSV) persisted throughout her life. The association of HSV infection with MAC and squamous cell carcinoma and that of peripheral T-cell lymphoma with Epstein-Barr virus is discussed in relation to her immunodeficiency.