Identification of non-tuberculous mycobacteria: 16S rRNA gene sequence analysis vs. conventional methods.

In a retrospective study, 45 clinical isolates of non-tuberculous mycobacteria were identified to the species level by biochemical profile, gas liquid chromatography and partial sequence analysis of 16S rRNA, and were found to represent 13 different species. The results of sequence analysis showed 100% identity with conventional tests for 34 isolates (76%) and could identify species such as M. bohemicum which are difficult to characterise with conventional methods. Most of the discrepant results for the remaining 11 isolates resulted in species of the same group of mycobacteria. Based on these findings. we concluded that direct sequence analysis of amplified 16S rRNA gene is a promising rapid and accurate method for species determination of non-tuberculous mycobacteria.

Septic arthritis caused by a gram-negative bacterium representing a new species related to the Bordetella-Alcaligenes complex.

A knee-joint exudate culture yielded on two occasions a gram-negative bacterium. Regular methods for speciation did not provide an identification. The infection was successfully treated with ciprofloxacin. The unknown isolate, CCUG 36768, was subjected to further investigation, including 16S rDNA sequencing, protein profiling, cellular fatty acid analysis, and various biochemical tests, in order to produce a species identification. The 1469 bp-long 16S rDNA sequence did not reveal identity with any known species sequence. CCUG 36768 clustered in a group of species, including Alcaligenes defragrans, Denitrobacter permanens, Taylorella equigenitalis, Alcaligenes faecalis, and four strains of Alcaligenes species without a specific species name. Bordetella species also showed a high degree of similarity with CCUG 36768. Protein profiling, cellular fatty acid analysis and computer-assisted analysis of biochemical profiles indicated similarity with Bordetella-Alcaligenes species, often close to B. holmesii and B. avium. API 20 NE indicated the profile of Moraxella species of poor identity. It is concluded that CCUG 36768 represents a new bacterial species of pathogenic potential in humans. It is related to the Bordetella-Alcaligenes group. Powerful new methods for speciation are available and it is recommended that unknown isolates from normally sterile sites be submitted for further analysis. Several isolates are required for the definition of new species.

Polymerase chain reaction for detection of Borrelia burgdorferi DNA in skin lesions of early and late Lyme borreliosis.

The aim of this study was to evaluate the polymerase chain reaction (PCR) as a diagnostic tool for Lyme borreliosis on large numbers of samples from clinically well-defined cases of early and late cutaneous borreliosis. Skin biopsy specimens from patients with erythema migrans and acrodermatitis chronica atrophicans were analysed blindly together with an equal number of control biopsies. Using two different dilutions of each DNA specimen increased the number of total positives detected. All of the 76 control biopsies were PCR negative. Biopsy specimens from 18 of 26 (69%) erythema migrans lesions and from 22 of 36 (61%) acrodermatitis chronica atrophicans lesions were PCR positive. Fourteen post-therapy biopsies from patients with acrodermatitis chronica atrophicans were all negative, supporting the opinion that antibiotic therapy is successful in this chronic manifestation of Lyme borreliosis.

Changes in Borrelia burgdorferi-specific serum IgG antibody levels in patients treated for acrodermatitis chronica atrophicans.

The kinetics of Borrelia burgdorferi-specific serum IgG antibody values in 74 patients treated for acrodermatitis chronica atrophicans was analysed by means of enzyme-linked immunosorbent assay. At the last clinical control, there had been no clinical signs of active infection. The serological follow-up time ranged from 12 months to 5 1/2 years (median 2 years and 1 month). In 68 (92%) of the 74 patients, a significant decrease of the specific antibody values was found within 3 years after the initiation of therapy. In 53 (72%) of the patients, this decrease was found within 15 months. Most of the patients remained seropositive during the follow-up period. The results show that a significant decline of the levels of serum IgG antibodies to Borrelia burgdorferi can be expected in the majority of patients who do not exhibit clinical evidence of persistent infection after antibiotic treatment of acrodermatitis chronica atrophicans.

Blood lymphocyte population following 131I treatment for hyperthyroidism.

A number of immunological parameters have been monitored for up to 6 weeks following 131I treatment for hyperthyroidism in Graves' disease. The aim was to examine whether this isotope treatment normalizes or further accentuates some immunological abnormalities which may be a manifestation of autoimmune reactions in these patients. It was confirmed that both the cellular composition and immunological reactivities of the patients' blood lymphocytes were abnormal before treatment. After 131I administration a slight lymphopenia occurred and the ratio between CD4 and CD8 positive T lymphocytes (helper-inducer/suppressor-cytotoxic), which was increased before treatment, increased further. Moreover, PWM-triggered IgM secretion in vitro was reduced by 50%. No other immunological parameters studied, such as secretion of other Ig classes, mitogenic responses of lymphocytes, and distribution of other lymphocyte subsets, changed to any detectable extent. It remains speculative whether the 131I-induced changes of the immune system may further accelerate the underlying autoimmune disease processes.

Comparison of four different serological methods for detection of antibodies to Borrelia burgdorferi in erythema migrans.

Three different enzyme-linked immunosorbent assays (ELISA) and Western blot were compared in regard to the detection of antibodies to Borrelia burgdorferi in sera from 100 patients with erythema migrans and from 100 controls. For IgG detection, a commercial indirect ELISA kit with flagellum antigen (flagellum ELISA) was significantly more sensitive than the routinely-used indirect ELISA with sonicated whole-cell antigen (sonicate ELISA) (p = 0.008). The difference in positivity in the IgM test was of borderline significance (p = 0.058). An IgM antibody-capture ELISA with sonicated whole-cell antigen (capture ELISA) was significantly more sensitive than either the IgM sonicate ELISA (p less than 0.001) or IgM flagellum ELISA (p less than 0.001). With the Western blot pattern chosen as the criterion for positivity, IgM Western blot was at least equal to IgM capture ELISA in terms of the number of positive erythema migrans sera, but a frequent discrepancy between these two tests was noted as to positivity in individual sera. IgG Western blot was considered to be of less value for the diagnosis of current disease due to a high occurrence of positivity among controls.

Influence of interferon on antibody production and viability of malignant cells from patients with multiple myeloma.

The influence of interferon (IFN) on antibody production and viability of malignant cells from patients with multiple myeloma was evaluated. Following incubation of bone marrow cells with IFN-alpha (5000 units/ml) for 7 days) a decreased production of monoclonal immunoglobin (mlg) was detected in all experiments except one. IFN induced a greater than 50% decrease in myeloma cell viability in 11 patients and a greater than or equal to 25% decrease in 4 patients, whereas in myeloma cells from 8 patients IFN had no or only minor effects. The observed effect was not due to an inhibition of proliferation since less than 5% of the myeloma cells were labeled with [3H]-thymidine during 7 d of culture. There was no statistically significant correlation between decreases in myeloma cell viability and effects on mlg production, exemplified by the fact that mlg production was decreased also in patients showing no sensitivity to IFN's cytotoxic action. Depletion of autologous T-cells, NK-cells and/or monocytes did not abrogate the effects observed. We conclude that IFN can reduce the viability of myeloma cells and the production of Ig from these cells and that the latter can be exerted without an antitumor effect.

Clinical signs and laboratory markers in predicting progression to AIDS in HIV-1 infected patients.

In a prospective longitudinal study 89 men with HIV-1 infection were observed for a mean time of 51 months with regard to clinical signs and laboratory findings predictive of progression to AIDS/opportunistic infection (OI). In a bivariate regression analysis the clinical signs showing a significant relation to AIDS development were: dermatitis of the face, yellow toe nail changes, hairly leukoplakia and oral candidiasis. The laboratory findings significantly associated with progression to AIDS were: decrease of the relative and absolute number of CD4 lymphocytes, decrease of the CD4/CD8 ratio, HIV p24 antigenaemia, lack of anti-HIV p24, elevated erythrocyte sedimentation rate, anaemia and elevated serum-beta-2-microglobulin. The relative number (%) of CD4 cells was found superior to the absolute number and the CD4/CD8 ratio. In a multivariate regression analysis decrease of CD4 lymphocytes and lack of anti-HIV p24 were independently associated with subsequent AIDS/OI development.

Immunological changes in primary HIV-1 infection.

Homosexual men with symptomatic primary HIV-1 infection displayed a pronounced lymphopaenia with significantly depressed numbers of CD3+, CD4+ and CD8+ cells and B cells during the first week of illness. Subsequently, the CD8+ cell counts rose in parallel with numbers of CD3+ cells, atypical lymphocytes and activated (CD38+ and HLA-Dr+) cells to attain maximal levels about a month following onset of illness. In contrast CD4+ and B cell numbers remained low for an extended period of time. Early signs of a host response included a transient appearance of interferon-alpha in the blood and raised levels of neopterin and beta 2-microglobulin (beta 2-M). Neither CD4+/CD8+ cell ratio nor beta 2-M resumed completely normal values during a follow-up period of 2 years. These findings shed some light on pathogenetic events during early HIV-1 infection and suggest that the infection, following the acute symptomatic stage, usually enters a stage of chronic active rather than latent infection.

Changes of the blood lymphocyte population following 32P treatment for polycythemia vera.

Orally administrated Na2 32PO4 mainly accumulates in bone marrow where it emits beta-particles which may damage cells. Previously, we showed that 32P treatment for polycythemia vera (PVC) increased the phytohemagglutinin reactivity and proportions of T cells in the blood. Now we have examined the effects of 32P treatment for PCV on natural killer (NK) and B-lymphocyte subsets which are considered to undergo their maturation in bone marrow. A mean isotope dose of 240 MBq given to 14 patients reduced the peripheral lymphocyte counts to 60% at 6 weeks. B cells and NK cells were reduced to the highest relative extent followed by HNK-1 cells and T cells. Although the proportion of NK cells was reduced to 50% there was no concomitant reduction of NK activity against K562 cells. Pokeweed mitogen-triggered secretion of IgM was significantly reduced, but not that of IgG or IgA. It is suggested that lymphocytes which mature in bone marrow may be affected to the highest extent by 32P treatment in PCV.

Elevated neopterin and beta 2-microglobulin levels in blood and cerebrospinal fluid occur early in HIV-1 infection.

Neopterin and beta 2-microglobulin (beta 2-M) concentrations in cerebrospinal fluid (CSF) and blood were measured in 56 individuals in various stages of HIV-1 infection. Elevated levels of neopterin as well as beta 2-M were found in the CSF of three patients with primary HIV-1 infection and also in subjects in the early stages of chronic HIV-1 infection, with the highest levels in HIV-1 isolation-positive people. There was a clear correlation between the concentrations of the two substances and the levels seemed to increase in parallel with progress of infection. A similar pattern was found in blood. Higher concentrations of neopterin and beta 2-M in CSF than in blood were found in patients with advanced dementia in particular. These findings indicate that the cellular immune system in the central nervous system (CNS) may be activated during the early stages of HIV-1 infection without concomitant overt neurological symptoms. The pathological processes in CNS and blood seem to develop in parallel rather than being restricted to one compartment.

Influence of adjuvant tamoxifen on blood lymphocytes.

The blood lymphocyte population was studied in 23 breast cancer patients treated with adjuvant tamoxifen for 1.5-2 years, and in an equal number of control patients. The size and cellular composition of the blood lymphocyte population, as assessed by monoclonal antibodies directed against various subsets, did not differ between the two patient groups. However, lymphocytes from the tamoxifen-treated patients exhibited a significantly lower NK activity against K562 cells. In contrast, the proliferative response of lymphocytes to ConA was significantly higher. These results indicate that tamoxifen may modulate the immune system.

Impact of adjuvant chemotherapy on spontaneous and poke week mitogen triggered immunoglobulin secretion of blood lymphocytes in operable breast cancer.

The objective of this investigation was to examine further the influence of postoperative adjuvant treatment with cyclophosphamide, methotrexate and 5-fluorouracil (CMF) on the immune system in breast cancer patients and to explore whether such changes are related to prognosis. The 12 CMF courses which were given for a period of 1 year resulted in a progressive lymphopenia. The relative spontaneous secretions of IgA and IgG in vitro increased 4- and 2-fold respectively after the first 3 courses, whereas IgM secretion was unaffected. IgA and IgG secretions in PWM stimulated cultures were not changed, whereas there was a sharp decrease of IgM. The CMF-induced changes of Ig-secretions were similar in patients who developed recurrent disease during a 4-6 year follow-up (n = 11) and those who remained clinically disease-free (n = 14). The results are discussed in relation to the immunopotentiation which may occur following treatment with relatively low doses of chemotherapeutic agents.

Elevated serum beta-2-microglobulin--a prognostic marker for development of AIDS among patients with persistent generalized lymphadenopathy.

For evaluation of its prognostic value, the level of serum beta-2-microglobulin was determined in early serum samples from 88 patients with persistent generalized lymphadenopathy in a prospective longitudinal study. Patients with serum beta-2-microglobulin greater than 2.6 mg/l were found to have a significantly higher risk of developing AIDS earlier when compared to patients with a lower level (p less than 0.001).

Changes of the blood lymphocyte subpopulations and their functions following 131I treatment for nodular goitre and 32P treatment for polycythemia vera.

The blood lymphocyte population was examined in 34 patients who were treated with 131I for toxic or atoxic nodular goitre. One to three doses of 300-550 MBq of 131I were administered at 1-week intervals. Lymphocyte counts were found to be significantly reduced at both 1 and 6 weeks after treatment. This decrease was accompanied by a changed composition of the lymphocyte subpopulations. The frequency of lymphocytes expressing membrane receptors for C'3 (EAC-rosette forming cells) was significantly reduced at 1 and 6 weeks following 131I administration. At 6 weeks there was a small but statistically significant increase of the frequency of T cells as identified by Leu 1 monoclonal antibodies. This was essentially due to an increased proportion of helper/inducer T cells as identified by Leu 3 monoclonals. 131I treatment also decreased the capacity of lymphocytes to secrete immunoglobulins (Ig) when stimulated with pokeweed mitogen (PWM). The greatest effect was observed for IgM. Secretion of IgG and IgA were less reduced. Mitogenic stimulations of lymphocytes with phytohemagglutinin (PHA) and concanavalin A were not significantly changed. It is concluded that these findings, with the exception of mitogen reactivity, are largely similar to those occurring following external radiation therapy for cancer. It is suggested that blood lymphocytes passing through the continuously irradiated gland are damaged mainly by beta-rays. The effect of 32P treatment on the blood lymphocyte population was examined in 16 patients with polycythemia vera. Before treatment the lymphocyte counts were within the normal range but the expression of certain membrane structures, as identified by monoclonal antibodies against total T cells (Leu 1 and 4), helper/inducer (Leu 3) and suppressor/cytotoxic T cells (Leu 2), were slightly decreased. Moreover, mitogenic responses of the lymphocytes to PHA and PWM-induced Ig secretion were impaired. Following a single oral dose of 32P (150-305 MBq), which normalized the production of erythrocytes and/or platelets, the blood lymphocyte counts were reduced by approximately 40 per cent 12 weeks after treatment. Examination of subsets demonstrated that the proportion of B-cells, as identified by B1 monoclonal antibodies, was decreased by the highest relative extent. On the other hand, lymphocytes expressing the above-mentioned T cell markers were somewhat increased. 32P treatment markedly increased PHA reactivity but it further reduced PWM-induced Ig secretion. The latter observation was in agreement with the finding that serum concentrations of Ig were reduced after treatment.

Effects of some prostaglandins and leukotrienes on lymphocytes, monocytes and their activity in vitro.

The effect of some prostaglandins (PGs) and leukotrienes (LTs) on lymphocytes and monocytes was tested in vitro. Overnight incubation with PGD2 reduces the expression of Leu-2 antigen (suppressor/cytotoxic phenotype) and of Fc receptors for IgG. PGA2, PGD2 and to a lesser extent PGE2 inhibit PHA reactivity of lymphocytes. LTB4 does not have any inhibitory activity. Preincubation of lymphocytes with PGD2 and PGE2 decreases their NK activity. LTB4, but not the PGs tested, stimulates monocyte metabolism as assessed by chemiluminescence.

Purified trichophytin: lymphocyte reactivity in vitro and trichophytin-induced suppression.

Three trichophytin preparations from different strains of Trichophyton mentagrophytes were produced according to the ethylene glycol method and assessed for their lymphocyte stimulation activity in vitro (LST). These trichophytin preparations showed significant variations in their effects on cord lymphocytes reactivity. The preparations also varied in their stimulation of lymphocytes from patients with dermatophytosis. Conclusions could be drawn about the immunological specificity, sensitivity and lymphocyte toxicity, which is important for the standardization of antigens. Furthermore, it was demonstrated that trichophytin-preincubated lymphocytes mediated a suppression of lymphocyte reactivity to this antigen.

T-cell helper activity and B-cell function of synovial and blood lymphocytes from patients with rheumatoid arthritis or other forms of chronic arthritis.

The helper effect of T cells on B-cell immunoglobulin (Ig) responses induced by pokeweed mitogen (PWM) or purified protein derivative of tuberculin (PPD) was studied in lymphocytes from synovial fluid (SF) and blood of nine patients with rheumatoid arthritis (RA) and eight patients with other forms of chronic arthritis. In PWM cultures the helper effect of SF T cells on Ig responses (IgG, IgM, IgA) of autologous and allogeneic blood B cells was lower than that of blood T cells (P less than 0.01). This decrease was more pronounced in patients with RA than in patients with non-RA. In PPD cultures no significant difference was found between the helper effect of SF T cells and blood T cells on the Ig responses of allogeneic blood B cells or on the IgG response of autologous blood B cells, whereas the helper effect of SF T cells on the IgM and IgA responses of autologous blood B cells was decreased. The Ig responses to PWM or PPD in cocultures of autologous blood B and T cells were not significantly different between patients and healthy controls. The PWM- and PPD-induced Ig responses of SF B cells were lower than those of blood B cells when cocultured with autologous blood T cells. SF B cells produced IgG but usually little IgM and IgA. Thus there was a dysfunction of SF B cells and of SF T cells in a PWM-driven system, but a fairly good helper function of SF T cells in a PPD-driven system.

Immunoregulatory function of T8 and T4 cells from synovial fluid and blood of patients with rheumatoid arthritis or other forms of chronic arthritis.

The suppressor effect of synovial fluid (SF) T8 cells and blood T8 cells on the pokeweed mitogen (PWM)-induced T4 cell-dependent immunoglobulin production of autologous blood B cells was studied in nine patients with chronic rheumatic diseases (six patients with rheumatoid arthritis (RA), one patient with juvenile RA, and two patients with other forms of chronic arthritis). The suppressor effect of SF T8 cells was of the same magnitude as that of equal numbers of blood T8 cells from patients and healthy controls. However, the relative number of T8 cells was higher among SF T cells than among blood T cells in several cases. Good synovial T8 cell suppression was also demonstrated in coculture experiments where SF T4 cells and B cells were used. In PPD (purified protein derivative of tuberculin)-stimulated cultures the suppressor effect of SF T8 cells as well as of blood T8 cells from patients and controls was lower than it was in PWM-stimulated cultures. In most patients SF T4 cells showed a much better PWM-induced helper function than did non-fractionated SF T cells. Thus the poor PWM induced helper effect of non fractionated synovial T cells was in some cases mainly due to the suppressor effect of T8 cells, whereas in some cases there was also a deficient helper function of synovial T4 cells.