Antibody response to bacteriophage hyaluronidase in acute glomerulonephritis after group A streptococcal infection.

In a test of the hypothesis that lysogeny of group A streptococci by a temperate bacteriophage might confer nephritogenicity, 283 sera from 69 patients were examined for IgG and IgM antibodies to M 49 streptococcal bacteriophage hyaluronidase. The IgG and IgM response to bacteriophage hyaluronidase was greatest in M 49 streptococci-infected individuals with nephritis, but M 49 streptococci-infected subjects without nephritis also had a greater immune response than did subjects infected with serotypes other than M 49. Although antibody to bacterial hyaluronidase was detected in all Streptococcus-infected groups, antibody to M 49 streptococcal bacteriophage hyaluronidase usually was found in only M 49 streptococci-infected patients. Although the greatest IgG and IgM antibody response to bacteriophage hyaluronidase can be demonstrated in individuals with glomerulonephritis, the antibody response does not indicate a direct relation of lysogeny and nephritis because subjects with and without nephritis after M 49 streptococcal infection all had a significant rise in antibody titer.

Lymphocyte subpopulations in rheumatic heart disease.

Monoclonal antibodies and indirect immunofluorescence techniques were used to compare the distribution of lymphocyte subpopulations of tonsil and peripheral blood from patients with rheumatic heart disease and age and socioeconomically matched patients undergoing tonsillectomy for chronic recurrent tonsillitis, but who had no evidence of rheumatic fever or rheumatic heart disease. The proportions of B cells (BA-1+), total T cells (Lyt-3), inducer/helper T cells (T4+) and cytotoxic/suppressor T cells (T8) were determined. No significant differences were apparent between rheumatic heart disease and control groups in resting cells from tonsils or blood. Cells undergoing proliferation in response to streptococcal blastogen A were identified by similar techniques. These tonsillar preparations from patients with rheumatic heart disease generated a smaller proportion of T8+ cultured cells and a greater T4/T8 ratio of cultured cells in response to group A streptococcal blastogen A than did nonrheumatic subjects.

Surface proteins in the transduction of groups A and G streptococci.

Four pairs of M+SOR+ and M-SOR- variants of group-A type-49 streptococci were compared as receptor strains in transduction of a streptomycin-resistance marker. The yield of transductants was 5-9-fold greater with the M-SOR- variants than with the corresponding M+SOR+ variants. Treatment of M+SOR+ variants of type-49 streptococci with trypsin enhanced the rate of transduction by 16-35-fold whereas trypsin treatment of corresponding M-SOR- variants resulted in minimal enhancement (5-fold or less). With trypsin treatment the numbers of transductants were approximately equal in pairs of M+SOR+ and M-SOR- variants. Enhanced transduction (10-26-fold) of streptomycin resistance was obtained by trypsin treatment of another seven M+SOR+ type-49 strains, of diverse phage subtypes and from various geographical locations. A wide range of enhancement (5-46-fold) was found in eight of nine M+ strains of group-A type-6 streptococci. With trypsin treatment, three of 10 transducible group-G strains showed enhanced transduction (10-13-fold) of a plasmid containing a determinant for erythromycin resistance. Transductional enhancement is proteolytic in nature, being enhanced by trypsin, chymotrypsin, papain, pronase and streptococcal proteinase. Although interference with phage adsorption by surface proteins would appear to be the most obvious explanation for these findings, further studies are required to define more clearly the mechanism of trypsin enhancement.

Enzyme-linked immunosorbent assay for identification and measurement of antibodies to group A streptococcal bacteriophage.

A sensitive enzyme immunoassay (ELISA) was developed to identify and measure antibodies to group A streptococcal bacteriophage hyaluronidase. With a purified preparation of bacteriophage hyaluronidase as the solid-phase antigen, the ELISA was shown to be as specific as and more sensitive than the standard bacteriophage neutralization test for measurement of antibody to bacteriophage. In rabbits immunized with bacteriophage, the ELISA detected antibody earlier than the neutralization assay (7 vs. 11 days) and was able to distinguish IgG and IgM class antibodies. A strong correlation was demonstrated between antibody titers measured by ELISA and bacteriophage neutralization (r = 0.88; P less than 0.001). Preliminary data using the ELISA, modified to measure human antibody to bacteriophage hyaluronidase, indicated that an antibody response of both IgG and IgM classes occurred in humans after group A streptococcal infection. This ELISA provided a sensitive method for detection and measurement of antibody to a specific bacteriophage antigen, which will be useful in the investigation of the role of bacteriophage in the pathogenesis of group A streptococcal infections.

Streptococcal toxins.

Few of the cellular components of group A streptococci appear to be directly toxic for animals or humans. Some preparations of M protein produce an immunotoxic effect on human platelets and neutrophils. Cell wall fragments produce a chronic multinodular inflammatory lesion of dermal connective tissue. The peptidoglycan component of cell walls has many of the biologic features of endotoxins. The exotoxins of group A streptococci include the erythrogenic toxins (pyrogenic exotoxins) and the cytolytic toxins (streptolysins S and O). The high prevalence of erythrogenic, toxin-producing strains is difficult to reconcile with the epidemiologic behavior of scarlet fever; the variations may be due to quantitative differences in toxin production or to a shift from the early scarlet fever-associated strains that produce A toxin to the currently prevalent strains that produce B and C toxins. Experiments with animals suggest that a positive Dick test and the rash of scarlet fever result not from a direct toxic effect but rather from enhancement by pyrogenic exotoxin(s) of acquired hypersensitivity to diverse streptococcal products. The mechanism of toxigenic phage conversion is not clear. The pyrogenic exotoxins are associated with the enhancement of endotoxin shock and a wide variety of other biologic properties. Streptolysin S is a nonantigenic polypeptide associated with various stabilizing carrier molecules. It lyses a wide range of mammalian cells, influences T lymphocyte functions, and is probably responsible for the leukotoxic property of group A streptococci. Rheumatic fever has been associated with a streptococcal outbreak due to a nonhemolytic (streptolysin S-negative) strain. Streptolysin O is an oxygen-labile (thiol-activated) cytolysin. It is inhibited by nonesterified cholesterol and binds to cholesterol in the membranes of mammalian cells and organelles, an interaction producing ring-like and C-shaped structures demonstrable by electron microscopy. Streptolysin O affects a number of leukocyte functions. It produces profound electrocardiographic changes in experimental animals and toxic effects on pulsating heart cells in tissue culture. The observation that rheumatic fever is not associated with infection of the skin due to group A streptococci has suggested that nonesterified cholesterol in the epidermis may inhibit a toxic effect of streptolysin O, an effect necessary for the development of rheumatic fever.

Type 49 Streptococcus pyogenes: phage subtypes as epidemiological markers in isolates from skin sepsis and acute glomerulonephritis.

Studies of group A, M type 49 streptococci from England, Trinidad and Alaska indicate that isolates of this serotype often differ with respect to phage subtype from one geographical area to another, but are generally homogeneous in one place at one time. The findings support the conclusion that acute glomerulonephritis can be associated with a variety of phage subtypes of M type 49 streptococci. In outbreaks of skin sepsis without nephritis in England, the phage subtypes of M type 49 streptococci isolated from skin lesions of meat handlers were the same as those recovered from skin lesions of non-meat handlers in the same community. The findings on the Trinidad isolates suggest that M type 49 streptococci of one phage subtype may persist in a population for 9 years and may result in a second outbreak of acute glomerulonephritis. In an Alaska Eskimo population in whom acute glomerulonephritis was occurring, most of the M type 49 isolates available for testing were of a single phage subtype. Equally prevalent in this population were group A streptococci that exhibited the same T antigen as the type 49 isolates but differed in their serum opacity reaction and phage subtype. This apparently related strain was not typable with available M antisera but showed functional evidence of M protein and is probably a new M type.

Factors influencing antibody responses to streptococcal M proteins in humans.

In a defined population of 174 children followed at one- to two-week intervals for 23 months, the development of M type-specific antibodies to four different types of group. A streptococci was assayed in 408 serum samples from 68 children who acquired one or more of these types in the upper respiratory tract and/or skin lesions. M type 6, a classic pharyngitis strain, was isolated almost exclusively from the upper respiratory tract. Acquisition of this type resulted in the highest percentage of responders without antibody to M protein and in the greatest magnitude of type-specific bactericidal activity as compared with M types 31, 49, and 52, which were commonly associated with pyoderma lesions. Factors that appeared to influence the M type-specific antibody response included the streptococcal type, the age of the child, the number of culture-positive visits, and the stage of the outbreak. The site(s) of isolation by itself and the serum opacity reaction of the infecting strain did not appear to be important determinants of the M type-specific antibody response.

Lisogenia em estreptococos do grupo A e avaliacao de meios de cultura para deteccao de bacteriofagos temperados obtidos desses microrganismos. / Lysogeny in group A streptococci and evaluation of culture media for detection of temperated bacteriophages obtained from these microorganisms

Cento e trinta e quatro amostras de estreptococos do grupo A foram examinadas quanto a ocorrencia de lisogenia. Utilizando-se quatro meios de cultura diferentes para o preparo do crescimento confluente da amostra indicadora em placas de agar, observamos uma porcentagem de deteccao de lisogenia de 14,2%. Dos 19 bacteriofagos obtidos, apenas 10 produziram lises na amostra indicadora preparada em quaisquer dos meios. Os restantes falharam para a producao de "plaque" em um ou mais meios. A taxa de lisogenia das amostras isoladas da orofaringe foi de 16,7% e 13,5% para as amostras obtidas de lesoes na pele. As porcentagens de amostras lisogenicas nos estreptococos M-tipaveis e nos Mnao tipaveis, para os microrganismos isolados da orofaringe, foram 16,7% e 20% respectivamente. Entretanto, para os estreptococos isolados de lesoes na pele, esta taxa foi de 24,2% para as amostras M-tipaveis e, 9,1% para as amostras M-nao tipaveis

Type-specific immunity and pharyngeal acquisition of group A Streptococcus.

A prospective study of spread of M-type 1, 2, 13, 14, 25 and 60 group A Streptococcus in 64 families in Qalyub, Egypt, in 1972-1974 showed that type-specific serum bactericidal antibody does not protect against pharyngeal acquisition of homologous organisms. The presence of type-specific antibody also does not appear to affect duration of carriage of the organism. Type-specific immunity must be mediated in another way, such as by local antibody or trough prevention of infection (as evidenced by a host response) following acquisition. This study also confirms the observations of others that administration of penicillin lowers the probability that a person who acquires group A Streptococcus will develop type-specific antibody.

Transduction of the genetic determinant for streptolysin S in group A streptococci.

The genetic determinant for streptolysin S production (SLS+) was successfully transduced to two naturally occurring nonhemolytic strains of group A streptococci (Streptococcus pyogenes), an M-type 18 strain associated with an outbreak of rheumatic fever and an M-negative variant of a type 49 strain isolated from a skin lesion. Attempts to transduce this determinant to a nonhemolytic M-type 68 strain and a nonhemolytic T-type 12 strain were not successful. Transduction was accomplished with a double temperature-sensitive mutant bacteriophage. Cellular antigenic characters and the phage sensitivity of the transductants remained unaltered. The donor strain also transduced streptomycin resistance well when the nonhemolytic type 49 strain was used as a recipient. There was no evidence of cotransduction of the determinants for streptolysin S and streptomycin resistance.

Functional alterations in non-T cells in rheumatic heart disease.

The mediation of the T cell lymphoproliferative response to streptococcal blastogen A by non-T mononuclear cells was studied in patients with rheumatic heart disease (RHD) and control subjects. Non-T cells are essential for T cell response to blastogen A. Non-T cells from RHD patients were less effective in enabling the T lymphocyte response to blastogen A than control non-T cells though no consistent difference was observed in the response to phytohaemagglutinin. The results suggest that a functional alteration is present in the non-T cells from RHD patients which might be related to the pathogenesis of the disease.

Characterization of the human cellular immune response to purified group A streptococcal blastogen A1.

The human mononuclear cell response to purified extracellular streptococcal protein, blastogen A, was compared to the response of these cells to PHA and tetanus antigen. Blastogen A induced peak uptake of thymidine during day 6 of tissue culture whereas PHA induced peak uptake during day 5 and tetanus during day 8. Like PHA, blastogen A transformed human umbilical cord lymphocytes and those of nonimmune animals. Also like PHA, blastogen A transformed primarily T lymphocytes. However, unlike PHA, the ability of T lymphocytes to respond to blastogen A was almost completely dependent on the presence of viable non-T lymphocytes. Monocytes were not as effective in facilitating the response to blastogen A as they were for PHA. Thus, blastogen A behaves most like a polyclonal T lymphocyte mitogen, although the degree of dependence of the transformation response on the presence of non-T lymphocytes is much greater than that of PHA.

Virulence factors in streptococci.

In man streptococcal septicaemia is most often associated with the streptococci of Lancefield's groups, A, B and D, with pneumococci, with Streptococcus milleri and with viridans streptococci. The specific determinants of the ability of streptococci to establish infection and to invade the blood stream are only partially understood. Even if fully comprehended, they would provide an incomplete picture of the factors responsible for the capacity of streptococci to produce a myriad of disease states the clinical expression of which include such diverse manifestations as acute sore throat, impetigo contagiosa, scarlet fever, erythema marginatum, St. Vitus dance, mitral stenosis, bloody urine and dental caries, to name only a few.